Prodrugs comprising an insulin linker conjugate

ABSTRACT

The present invention relates to a prodrug or a pharmaceutically acceptable salt thereof comprising an insulin linker conjugate D-L, wherein D represents the insulin moiety; and -L is a non-biologically active linker moiety -L 1  represented by formula (I), 
     
       
         
         
             
             
         
       
     
     wherein the dashed line indicates the attachment to one of the amino groups of the insulin by forming an amide bond. The invention further relates to pharmaceutical compositions comprising said prodrugs as well as their use as a medicament for treating or preventing diseases or disorders which can be treated by insulin.

The present invention relates to prodrugs, pharmaceutical compositionscomprising said prodrugs as well as their use as a medicament fortreating or preventing diseases or disorders which can be treated byinsulin.

Insulin therapy is characterized by a high need for keeping the insulindrug release within very strict levels as the therapeutic window isnarrow, and the adverse effects of hyperinsulinemia can potentially belife threatening. Numerous insulin preparations have beencommercialized, with different action profiles to suit specific needs ofthe diabetic population. Fast acting insulin analogs are administeredjust before meals, in order to control the peak in plasma glucosefollowing food ingestion, whereas long acting insulin analogs aretypically given once or twice a day to provide a steady basal insulinlevel.

Therefore, there is a clear need for novel long acting preparations ofinsulin, that continuously release insulin throughout the entire periodbetween administrations.

WO-A 2006/003014 describes a hydrogel capable of releasing insulin withthe possibility of reduced dosing frequency as compared to standarddaily basal insulin injections. However, the insulin is released at arate too fast for ensuring strict insulinotropic control for periodsextending 2 days. In fact the insulin is released with a half life ofapproximately 30 hours, meaning that the prodrug must be administered atleast every 30 hours in order for the peak to trough ratio to be below 2at steady state.

The concept of preparing a reversible polymer prodrug conjugate ofinsulin has been explored by Shechter et al. and described in scientificarticles and patent applications (e.g. European Journal of Pharmaceuticsand Biopharmaceutics 2008(70), 19-28 and WO-A 2004/089280). The insulinis conjugated to a 40 kDa PEG polymer through a fluorenyl-linker.Hydrolysis of said linker molecule releases insulin with a half life ofapproximately 30 hours, meaning that the prodrug must be administered atleast every 30 hours in order for the peak to trough ratio to be below 2at steady state.

Other attempts of reducing the insulin dosing frequency have been made.Hinds et al., Journal of Controlled Release, 2005 (104), 447-460,describe a method of producing a once weekly insulin, by firstpermanently PEGylating the insulin molecule and then subsequentlymicroencapsulating the PEGylated insulin in PLGA microparticles. In thiscase, the insulin was subjected to substantial structural modificationthrough permanent modification by a high molecular weight polymerentity. Such high molecular weight modified insulins may exhibit reducedefficacy by diminished receptor binding and may also exhibit injectionsite reactions such as lipoatrophy due to the extended presence of highconcentrations of the high molecular weight insulin in the subcutaneoustissue. Furthermore, such PEGylated insulins will exhibit a lowerdistribution volume, which is of particular disadvantage in thetreatment of diabetes.

Nevertheless, PEGylation of insulin apparently serves to protect thepeptide from deterioration in the PLGA polymer formulation. The effectof PEGylation to protect peptides from acylation in a degrading PLGAformulation was demonstrated for octreotide by D. H. Na et al., AAPSPharmSciTech 2003, 4 (4) Article 72.

PLGA encapsulation of proteins has been shown to cause side reactions ofthe polymer esters with peptide or protein amino groups. Lactic acidacylation products have been observed after exposure of the formulationsto buffered solutions at neutral pH (G. Zhu et al., Nature Biotechnology18 (2000) 52-57; A. J. Domb et al., Pharm. Res. 11 (1994) 865-868; A.Lucke et al., Pharm. Res. 19 (2002) 175-181).

Specifically for insulin, detrimental effects of polymer formulationshave been demonstrated by P. G. Shao et al., Pharm. Dev. Technol. 4(1999) 633-642 (see also P. G. Shao et al., Pharm. Dev. Technol. 5(2000)1-9).

Furthermore, insulin is known to readily undergo side reactions that arerelated to the presence of three disulfide bridges in the molecule. Forinstance, insulin may be split into A and B chains by disulfide bondcleavage or dimers or oligomers may be formed due to disulfideinterchange reactions. Such disulfide reshuffling is particularlylikely, if insulin molecules are forced into close contact in a randomway. This intrinsic lability of the insulin molecule has significantlyhampered progress in long-acting depot development and prevented the useof other polymer formulations where insulin is encapsulated in a waysimilar to an amorphous precipitate which is well known to give rise tovarious degradation products arising from extensive disulfide exchange.

Therefore the challenge remains to develop long-acting insulin withoutcompromising the insulin pharmacodynamics by permanent attachment of ahigh molecular weight entity or by causing structural damage to themolecule while during its presence in the depot.

Thus an object of the present invention is to provide an insulincontaining prodrug that meets at least partially the above requirements.

The object is achieved by a prodrug or a pharmaceutically acceptablesalt thereof comprising an insulin linker conjugate D-L, wherein

D represents the insulin moiety; and

-L is a non-biologically active linker moiety -L¹ represented by formula(I),

wherein the dashed line indicates the attachment to one of the aminogroups of the insulin by forming an amide bond;

X is C(R³R^(3a)); or N(R³);

R^(1a), R^(3a) are independently selected from the group consisting H,NH(R^(2b)), N(R^(2b))C(O)R⁴ and C₁₋₄ alkyl;

R¹, R², R^(2a), R^(2b), R³, R⁴ are independently selected from the groupconsisting of H and C₁₋₄ alkyl,

wherein L¹ is substituted with one L²-Z and optionally furthersubstituted, provided that the hydrogen marked with the asterisk informula (I) is not replaced by a substituent and wherein

L² is a single chemical bond or a spacer; and

Z is a hydrogel.

It was now surprisingly discovered, that a prodrug of the presentinvention may provide insulin release from a subcutaneous depot instructurally intact form over time periods of at least 2 days betweenadministrations. As a further advantage structural integrity of thereleased insulin may be provided by a well-hydrated polymer matrixminimizing intermolecular contact of insulin molecules and sustainedrelease may be enabled by means of a self-cleaving prodrug linkerbetween the insulin and the polymer matrix.

Thus it should be possible to administer insulin in form of a prodrug ofthe present invention less frequently than current long acting insulins.Further advantages should be a small peak to trough ratio, which greatlyreduce the risk of hypoglycemic episodes. This may help patients toreduce the frequency of injections, while being able to maintain optimalcontrol the plasma levels of insulin and consequently blood glucose.

“Insulin” according to the present invention means recombinant humaninsulin, lantus, insulin glargine, insulin detemir, insulin glulisine,insulin aspart, insulin lispro, insulin conjugated tolow-molecular-weight PEG. Low-molecular-weight PEG has a molecularweight smaller than 10 kDa.

Insulin bound to a non-biologically active linker is referred to as“insulin moiety”.

By “insulin analogue” as used herein is meant a polypeptide which has amolecular structure which formally can be derived from the structure ofa naturally occurring insulin, for example that of human insulin, bydeleting and/or exchanging at least one amino acid residue occurring inthe naturally occurring insulin and/or adding at least one amino acidresidue. The added and/or exchanged amino acid residues can either becodable amino acid residues or other naturally occurring residues orpurely synthetic amino acid residues.

The insulin analogues may be such wherein position 28 of the B chain maybe modified from the natural Pro residue to one of Asp, Lys, or Iie. Inanother aspect Lys at position B29 is modified to Pro. Also, Asn atposition A21 may be modified to Ala, Gln, Glu, Gly, His, Iie, Leu, Met,Ser, Thr, Trp, Tyr or Val, in particular to Gly, Ala, Ser, or Thr andpreferably to Gly. Furthermore, Asn at position B3 may be modified toLys or Asp. Further examples of insulin analogues are des(B30) humaninsulin; des(B30) human insulin analogues; insulin analogues whereinPheB1 has been deleted; insulin analogues wherein the A-chain and/or theB-chain have an N-terminal extension and insulin analogues wherein theA-chain and/or the B-chain have a C-terminal extension. Thus one or twoArg may be added to position B1.

With desB30 insulin”, “desB30 human insulin” is meant a natural insulinor an analogue thereof lacking the B30 amino acid residue. Similarly,“desB29desB30 insulin” or desB29desB30 human insulin” means a naturalinsulin or an analogue thereof lacking the B29 and B30 amino acidresidues.

With “B1”, “A1” etc. is meant the amino acid residue at position 1 inthe B-chain of insulin (counted from the N-terminal end) and the aminoacid residue at position 1 in the A-chain of insulin (counted from theN-terminal end), respectively. The amino acid residue in a specificposition may also be denoted as e.g. PheB1 which means that the aminoacid residue at position B1 is a phenylalanine residue.

“Non-biologically active linker” means a linker which does not show thepharmacological effects of the drug derived from the biologically activeagent.

“Protective groups” refers to a moiety which temporarily protects achemical functional group of a molecule during synthesis to obtainchemoselectivity in subsequent chemical reactions. Protective groups foralcohols are, for example, benzyl and trityl, protective groups foramines are, for example, tert-butyloxycarbonyl,9-fluorenylmethyloxycarbonyl and benzyl and for thiols examples ofprotective groups are 2,4,6-trimethoxybenzyl, phenylthiomethyl,acetamidomethyl, p-methoxybenzyloxycarbonyl, tert-butylthio,triphenylmethyl, 3-nitro-2-pyridylthio, 4-methyltrityl.

“Protected functional groups” means a chemical functional groupprotected by a protective group.

“Acylating agent” means a moiety of the structure R—(C═O)—, providingthe acyl group in an acylation reaction, optionally connected to aleaving group, such as acid chloride, N-hydroxy succinimide,pentafluorphenol and para-nitrophenol.

“Alkyl” means a straight-chain or branched carbon chain. Each hydrogenof an alkyl carbon may be replaced by a substituent.

“Aryl” refers to any substituent derived from a monocyclic or polycyclicor fused aromatic ring, including heterocyclic rings, e.g. phenyl,thiophene, indolyl, napthyl, pyridyl, which may optionally be furthersubstituted.

“Acyl” means a chemical functional group of the structure R—(C═O)—,wherein R is an alkyl or aryl.

“C₁₋₄ alkyl” means an alkyl chain having 1-4 carbon atoms, e.g. ifpresent at the end of a molecule: methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl tert-butyl, or e.g. —CH₂—, —CH₂—CH₂—,—CH(CH₃)—, —CH₂—CH₂—CH₂—, —CH(C₂H₅)—, —C(CH₃)₂—, when two moieties of amolecule are linked by the alkyl group. Each hydrogen of a C₁₋₄ alkylcarbon may be replaced by a substituent.

“C₁₋₆ alkyl” means an alkyl chain having 1-6 carbon atoms, e.g. ifpresent at the end of a molecule: C₁₋₄ alkyl, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl; tert-butyl, n-pentyl, n-hexyl,or e.g. —CH₂—, —CH₂—CH₂—, —CH(CH₃)—, —CH₂—CH₂—CH₂—, —CH(C₂H₅)—,—C(CH₃)₂—, when two moieties of a molecule are linked by the alkylgroup. Each hydrogen of a C₁₋₆ alkyl carbon may be replaced by asubstituent.

Accordingly, “C₁₋₁₈ alkyl” means an alkyl chain having 1 to 18 carbonatoms and “C₈₋₁₈ alkyl” means an alkyl chain having 8 to 18 carbonatoms. Accordingly, “C₁₋₅₀ alkyl” means an alkyl chain having 1 to 50carbon atoms.

“C₂₋₅₀ alkenyl” means a branched or unbranched alkenyl chain having 2 to50 carbon atoms, e.g. if present at the end of a molecule: —CH═CH₂,—CH═CH—CH₃, —CH₂—CH═CH₂, —CH═CH—CH₂—CH₃, —CH═CH—CH═CH₂, or e.g. —CH═CH—,when two moieties of a molecule are linked by the alkenyl group. Eachhydrogen of a C₂₋₅₀ alkenyl carbon may be replaced by a substituent asfurther specified. Accordingly, the term “alkenyl” relates to a carbonchain with at least one carbon carbon double bond. Optionally, one ormore triple bonds may occur.

“C₂₋₅₀ alkynyl” means a branched or unbranched alkynyl chain having 2 to50 carbon atoms, e.g. if present at the end of a molecule: —C≡CH,—CH₂—C≡CH, CH₂—CH₂—C≡CH, CH₂—C≡C—CH₃, or e.g. —C≡C— when two moieties ofa molecule are linked by the alkynyl group. Each hydrogen of a C₂₋₅₀alkynyl carbon may be replaced by a substituent as further specified.Accordingly, the term “alkynyl” relates to a carbon chain with at lestone carbon carbon triple bond. Optionally, one or more double bonds mayoccur.

“C₃₋₇ cycloalkyl” or “C₃₋₇ cycloalkyl ring” means a cyclic alkyl chainhaving 3 to 7 carbon atoms, which may have carbon-carbon double bondsbeing at least partially saturated, e.g. cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl. Each hydrogen of acycloalkyl carbon may be replaced by a substituent. The term “C₃₋₇cycloalkyl” or “C₃₋₇ cycloalkyl ring” also includes bridged bicycleslike norbonane or norbonene. Accordingly, “C₃₋₅ cycloalkyl” means acycloalkyl having 3 to 5 carbon atoms.

Accordingly, “C₃₋₁₀ cycloalkyl” means a cyclic alkyl having 3 to 10carbon atoms, e.g. C₃₋₇ cycloalkyl; cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl,cyclononyl, cyclodecyl. The term “C₃₋₁₀ cycloalkyl” also includes atleast partially saturated carbomono- and -bicycles.

“Halogen” means fluoro, chloro, bromo or iodo. It is generally preferredthat halogen is fluoro or chloro.

“4 to 7 membered heterocyclyl” or “4 to 7 membered heterocycle” means aring with 4, 5, 6 or 7 ring atoms that may contain up to the maximumnumber of double bonds (aromatic or non-aromatic ring which is fully,partially or un-saturated) wherein at least one ring atom up to 4 ringatoms are replaced by a heteroatom selected from the group consisting ofsulfur (including —S(O)—, —S(O)₂—), oxygen and nitrogen (including═N(O)—) and wherein the ring is linked to the rest of the molecule via acarbon or nitrogen atom. Examples for a 4 to 7 membered heterocycles areazetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline,imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline,isoxazole, isoxazoline, thiazole, thiazoline, isothiazole,isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran,tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine,oxazolidine, isoxazolidine, thiazolidine, isothiazolidine,thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran,imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine,piperidine, morpholine, tetrazole, triazole, triazolidine,tetrazolidine, diazepane, azepine or homopiperazine.

“9 to 11 membered heterobicyclyl” or “9 to 11 membered heterobicycle”means a heterocyclic system of two rings with 9 to 11 ring atoms, whereat least one ring atom is shared by both rings and that may contain upto the maximum number of double bonds (aromatic or non-aromatic ringwhich is fully, partially or un-saturated) wherein at least one ringatom up to 6 ring atoms are replaced by a heteroatom selected from thegroup consisting of sulfur (including —S(O)—, —S(O)₂—), oxygen andnitrogen (including ═N(O)—) and wherein the ring is linked to the restof the molecule via a carbon or nitrogen atom. Examples for a 9 to 11membered heterobicycle are indole, indoline, benzofuran, benzothiophene,benzoxazole, benzisoxazole, benzothiazole, benzisothiazole,benzimidazole, benzimidazoline, quinoline, quinazoline,dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline,decahydroquinoline, isoquinoline, decahydroisoquinoline,tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine orpteridine. The term 9 to 11 membered heterobicycle also includes spirostructures of two rings like 1,4-dioxa-8-azaspiro[4.5]decane or bridgedheterocycles like 8-aza-bicyclo[3.2.1]octane.

In case the insulin prodrugs comprising the compounds according toformula (I) contain one or more acidic or basic groups, the inventionalso comprises their corresponding pharmaceutically or toxicologicallyacceptable salts, in particular their pharmaceutically utilizable salts.Thus, the insulin prodrugs comprising the compounds of the formula (I)which contain acidic groups can be used according to the invention, forexample, as alkali metal salts, alkaline earth metal salts or asammonium salts. More precise examples of such salts include sodiumsalts, potassium salts, calcium salts, magnesium salts or salts withammonia or organic amines such as, for example, ethylamine,ethanolamine, triethanolamine or amino acids. Insulin prodrugscomprising the compounds of the formula (I) which contain one or morebasic groups, i.e. groups which can be protonated, can be present andcan be used according to the invention in the form of their additionsalts with inorganic or organic acids. Examples for suitable acidsinclude hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuricacid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid,lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid,pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelicacid, fumaric acid, maleic acid, malic acid, sulfaminic acid,phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid,citric acid, adipic acid, and other acids known to the person skilled inthe art. If the insulin prodrugs comprising the compounds of the formula(I) simultaneously contain acidic and basic groups in the molecule, theinvention also includes, in addition to the salt forms mentioned, innersalts or betaines (zwitterions). The respective salts according toinsulin prodrugs comprising the formula (I) can be obtained by customarymethods which are known to the person skilled in the art like, forexample by contacting these with an organic or inorganic acid or base ina solvent or dispersant, or by anion exchange or cation exchange withother salts. The present invention also includes all salts of theinsulin prodrugs comprising the compounds of the formula (I) which,owing to low physiological compatibility, are not directly suitable foruse in pharmaceuticals but which can be used, for example, asintermediates for chemical reactions or for the preparation ofpharmaceutically acceptable salts.

To enhance physicochemical or pharmacokinetic properties of a drug, suchas insulin, in vivo, such drug can be conjugated with a carrier. If thedrug is transiently bound to a carrier and/or a linker, such systems arecommonly assigned as carrier-linked prodrugs. According to thedefinitions provided by IUPAC (as given underhttp://www.chem.qmul.ac.uk/iupac.medchem, accessed on Jul. 22, 2009), acarrier-linked prodrug is a prodrug that contains a temporary linkage ofa given active substance with a transient carrier group that producesimproved physicochemical or pharmacokinetic properties and that can beeasily removed in vivo, usually by a hydrolytic cleavage.

The linkers employed in such carrier-linked prodrugs are transient,meaning that they are non-enzymatically hydrolytically degradable(cleavable) under physiological conditions (aqueous buffer at pH 7.4,37° C.) with half-lives ranging from, for example, one hour to threemonths.

Suitable carriers are polymers and can either be directly conjugated tothe linker or via a non-cleavable spacer. The term “insulin hydrogelprodrug” refers to carrier-linked prodrugs of insulin, wherein thecarrier is a hydrogel. The terms “hydrogel prodrug” and “hydrogel-linkedprodrug” refer to prodrugs of biologically active agents transientlylinked to a hydrogel and are used synonymously. A “hydrogel” may bedefined as a three-dimensional, hydrophilic or amphiphilic polymericnetwork capable of taking up large quantities of water. The networks arecomposed of homopolymers or copolymers, are insoluble due to thepresence of covalent chemical or physical (ionic, hydrophobicinteractions, entanglements) crosslinks. The crosslinks provide thenetwork structure and physical integrity. Hydrogels exhibit athermodynamic compatibility with water which allows them to swell inaqueous media. The chains of the network are connected in such a fashionthat pores exist and that a substantial fraction of these pores are ofdimensions between 1 nm and 1000 nm.

“Free form” of a drug refers to the drug in its unmodified,pharmacologically active form, such as after being released from apolymer conjugate.

The terms “drug”, “biologically active molecule”, “biologically activemoiety”, “biologically active agent”, “active agent”, and the like meanany substance which can affect any physical or biochemical properties ofa biological organism, including but not limited to viruses, bacteria,fungi, plants, animals, and humans. In particular, as used herein,biologically active molecules include any substance intended fordiagnosis, cure, mitigation, treatment, or prevention of disease inhumans or other animals, or to otherwise enhance physical or mentalwell-being of humans or animals. Specifically, the terms “drug”,“biologically active molecule”, “biologically active moiety”,“biologically active agent”, “active agent”, and the like refer toinsulin.

A “therapeutically effective amount” of insulin as used herein means anamount sufficient to cure, alleviate or partially arrest the clinicalmanifestations of a given disease and its complications. An amountadequate to accomplish this is defined as “therapeutically effectiveamount”. Effective amounts for each purpose will depend on the severityof the disease or injury as well as the weight and general state of thesubject. It will be understood that determining an appropriate dosagemay be achieved using routine experimentation, by constructing a matrixof values and testing different points in the matrix, which is allwithin the ordinary skills of a trained physician.

“Stable” and “stability” means that within the indicated storage timethe hydrogel conjugates remain conjugated and do not hydrolyze to asubstantial extent and exhibit an acceptable impurity profile relatingto insulin. To be considered stable, the composition contains less than5% of the drug in its free form.

The term “pharmaceutically acceptable” means approved by a regulatoryagency such as the EMEA (Europe) and/or the FDA (US) and/or any othernational regulatory agency for use in animals, preferably in humans.

“Pharmaceutical composition” or “composition” means one or more activeingredients, and one or more inert ingredients, as well as any productwhich results, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions of the present invention encompass anycomposition made by admixing a compound of the present invention and apharmaceutically acceptable excipient (pharmaceutically acceptablecarrier).

“Dry composition” means that the insulin hydrogel prodrug composition isprovided in a dry form in a container. Suitable methods for drying arespray-drying and lyophilization (freeze-drying). Such dry composition ofinsulin hydrogel prodrug has a residual water content of a maximum of10%, preferably less than 5% and more preferably less than 2%(determined according to Karl Fischer). The preferred method of dryingis lyophilization. “Lyophilized composition” means that the insulinhydrogel polymer prodrug composition was first frozen and subsequentlysubjected to water reduction by means of reduced pressure. Thisterminology does not exclude additional drying steps which occur in themanufacturing process prior to filling the composition into the finalcontainer.

“Lyophilization” (freeze-drying) is a dehydration process, characterizedby freezing a composition and then reducing the surrounding pressureand, optionally, adding heat to allow the frozen water in thecomposition to sublime directly from the solid phase to gas. Typically,the sublimed water is collected by desublimation.

“Reconstitution” means the addition of a liquid to bring back theoriginal form of a composition.

“Reconstitution solution” refers to the liquid used to reconstitute thedry composition of a insulin hydrogel prodrug prior to administration toa patient in need thereof.

“Container” means any container in which the insulin hydrogel prodrugcomposition is comprised and can be stored until reconstitution.

“Buffer” or “buffering agent” refers to chemical compounds that maintainthe pH in a desired range. Physiologically tolerated buffers are, forexample, sodium phosphate, succinate, histidine, bicarbonate, citrateand acetate, sulphate, nitrate, chloride, pyruvate. Antacids such asMg(OH)₂ or ZnCO₃ may be also used. Buffering capacity may be adjusted tomatch the conditions most sensitive to pH stability.

“Excipients” refers to compounds administered together with thetherapeutic agent, for example, buffering agents, isotonicity modifiers,preservatives, stabilizers, anti-adsorption agents, oxidation protectionagents, or other auxiliary agents. However, in some cases, one excipientmay have dual or triple functions.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest, significantly prevents or reduces chemical and/or physicalinstability of the protein upon drying in general and especially duringlyophilization and subsequent storage. Exemplary lyoprotectants includesugars, such as sucrose or trehalose; amino acids such as arginine,glycine, glutamate or histidine; methylamines such as betaine; lyotropicsalts such as magnesium sulfate; polyols such as trihydric or highersugar alcohols, e.g. glycerin, erythritol, glycerol, arabitol, xylitol,sorbitol, and mannitol; ethylene glycol; propylene glycol; polyethyleneglycol; pluronics; hydroxyalkyl starches, e.g. hydroxyethyl starch(HES), and combinations thereof.

“Surfactant” refers to wetting agents that lower the surface tension ofa liquid.

“Isotonicity modifiers” refer to compounds which minimize pain that canresult from cell damage due to osmotic pressure differences at theinjection depot.

The term “stabilizers” refers to compounds used to stabilize the polymerprodrug. Stabilisation is achieved by strengthening of theprotein-stabilising forces, by destabilisation of the denatured state,or by direct binding of excipients to the protein.

“Anti-adsorption agents” refers to mainly ionic or non-ionic surfactantsor other proteins or soluble polymers used to coat or adsorbcompetitively to the inner surface of the composition's container.

Chosen concentration and type of excipient depends on the effect to beavoided but typically a monolayer of surfactant is formed at theinterface just above the CMC value.

“Oxidation protection agents” refers to antioxidants such as ascorbicacid, ectoine, glutathione, methionine, monothioglycerol, morin,polyethylenimine (PEI), propyl gallate, vitamin E, chelating agents suchas citric acid, EDTA, hexaphosphate, thioglycolic acid.

“Antimicrobial” refers to a chemical substance that kills or inhibitsthe growth of microorganisms, such as bacteria, fungi, yeasts,protozoans and/or destroys viruses.

“Sealing a container” means that the container is closed in such waythat it is airtight, allowing no gas exchange between the outside andthe inside and keeping the content sterile.

The term “reagent” or “precursor” refers to an intermediate or startingmaterial used in the assembly process leading to a prodrug of thepresent invention.

The term “chemical functional group” refers to carboxylic acid andactivated derivatives, amino, maleimide, thiol and derivatives, sulfonicacid and derivatives, carbonate and derivatives, carbamate andderivatives, hydroxyl, aldehyde, ketone, hydrazine, isocyanate,isothiocyanate, phosphoric acid and derivatives, phosphonic acid andderivatives, haloacetyl, alkyl halides, acryloyl and other alpha-betaunsaturated michael acceptors, arylating agents like aryl fluorides,hydroxylamine, disulfides like pyridyl disulfide, vinyl sulfone, vinylketone, diazoalkanes, diazoacetyl compounds, oxirane, and aziridine.

If a chemical functional group is coupled to another chemical functionalgroup, the resulting chemical structure is referred to as “linkage”. Forexample, the reaction of an amine group with a carboxyl group results inan amide linkage.

“Reactive functional groups” are chemical functional groups of thebackbone moiety, which are connected to the hyperbranched moiety.

“Functional group” is the collective term used for “reactive functionalgroup”, “degradable interconnected functional group”, or “conjugatefunctional group”.

A “degradable interconnected functional group” is a linkage comprising abiodegradable bond which on one side is connected to a spacer moietyconnected to a backbone moiety and on the other side is connected to thecrosslinking moiety. The terms “degradable interconnected functionalgroup”, “biodegradable interconnected functional group”, “interconnectedbiodegradable functional group” and “interconnected functional group”are used synonymously.

The terms “blocking group” or “capping group” are used synonymously andrefer to moieties which are irreversibly connected to reactivefunctional groups to render them incapable of reacting with for examplechemical functional groups.

The terms “protecting group” or “protective group” refers to a moietywhich is reversibly connected to reactive functional groups to renderthem incapable of reacting with for example other chemical functionalgroups.

The term “interconnectable functional group” refers to chemicalfunctional groups, which participate in a radical polymerizationreaction and are part of the crosslinker reagent or the backbonereagent.

The term “polymerizable functional group” refers to chemical functionalgroups, which participate in a ligation-type polymerization reaction andare part of the crosslinker reagent and the backbone reagent.

A backbone moiety may comprise a spacer moiety which at one end isconnected to the backbone moiety and on the other side to thecrosslinking moiety.

The term “derivatives” refers to chemical functional groups suitablysubstituted with protecting and/or activation groups or to activatedforms of a corresponding chemical functional group which are known tothe person skilled in the art. For example, activated forms of carboxylgroups include but are not limited to active esters, such assuccinimidyl ester, benzotriazyl ester, nitrophenyl ester,pentafluorophenyl ester, azabenzotriazyl ester, acyl halogenides, mixedor symmetrical anhydrides, acyl imidazole.

The term “non-enzymatically cleavable linker” refers to linkers that arehydrolytically degradable under physiological conditions withoutenzymatic activity.

“Non-biologically active linker” means a linker which does not show thepharmacological effects of the drug (D-H) derived from the biologicallyactive moiety.

The terms “spacer”, “spacer group”, “spacer molecule”, and “spacermoiety” are used interchangeably and if used to describe a moietypresent in the hydrogel carrier of the invention, refer to any moietysuitable for connecting two moieties, such as C₁₋₅₀ alkyl, C₂₋₅₀ alkenylor C₂₋₅₀ alkinyl, which fragment is optionally interrupted by one ormore groups selected from —NH—, —N(C₁₋₄ alkyl)-, —O—, —S—, —C(O)—,—C(O)NH—, —C(O)N(C₁₋₄ alkyl)-, —O—C(O)—, —S(O)—, —S(O)₂—, 4 to 7membered heterocyclyl, phenyl or naphthyl.

The terms “terminal”, “terminus” or “distal end” refer to the positionof a functional group or linkage within a molecule or moiety, wherebysuch functional group may be a chemical functional group and the linkagemay be a degradable or permanent linkage, characterized by being locatedadjacent to or within a linkage between two moieties or at the end of anoligomeric or polymeric chain.

The phrases “in bound form” or “moiety” refer to sub-structures whichare part of a larger molecule. The phrase “in bound form” is used tosimplify reference to moieties by naming or listing reagents, startingmaterials or hypothetical starting materials well known in the art, andwhereby “in bound form” means that for example one or more hydrogenradicals (—H), or one or more activating or protecting groups present inthe reagents or starting materials are not present in the moiety.

It is understood that all reagents and moieties comprising polymericmoieties refer to macromolecular entities known to exhibit variabilitieswith respect to molecular weight, chain lengths or degree ofpolymerization, or the number of functional groups. Structures shown forbackbone reagents, backbone moieties, crosslinker reagents, andcrosslinker moieties are thus only representative examples.

A reagent or moiety may be linear or branched. If the reagent or moietyhas two terminal groups, it is referred to as a linear reagent ormoiety. If the reagent or moiety has more than two terminal groups, itis considered to be a branched or multi-functional reagent or moiety.

The term “poly(ethylene glycol) based polymeric chain” or “PEG basedchain” refers to an oligo- or polymeric molecular chain.

Preferably, such poly(ethylene glycol) based polymeric chain isconnected to a branching core, it is a linear poly(ethylene glycol)chain, of which one terminus is connected to the branching core and theother to a hyperbranched dendritic moiety. It is understood that aPEG-based chain may be terminated or interrupted by alkyl or aryl groupsoptionally substituted with heteroatoms and chemical functional groups.

If the term “poly(ethylene glycol) based polymeric chain” is used inreference to a crosslinker reagent, it refers to a crosslinker moiety orchain comprising at least 20 weight % ethylene glycol moieties.

Preferably, in formula (I) R² is replaced by L²-Z.

Preferably, in formula (I) R¹ is replaced by L²-Z.

Preferably, in formula (I) X is N(R³).

Preferably, in formula (I) X is C(R³R^(3a)) and R^(3a) isN(R^(2b))C(O)R⁴.

Preferably, in formula (I) X is C(R³R^(3a)) and R^(3a) is replaced byL²-Z.

Preferably, X is C(R³R^(3a)), R^(3a) is N(R^(2b))-L²-Z.

Preferred prodrugs of the present invention comprise an insulin linkerconjugate D-L, wherein L² of formula (I) is represented by formulae(Ia), (Ib), (Ic) or (Id):

wherein R¹, R^(1a), R², R^(2a), R^(2b), R³, R⁴, L², Z have the meaningas indicated herein and wherein L¹ is optionally further substituted,provided that the hydrogen marked with the asterisk in formula (Ia) to(Id) is not replaced by a substituent.

Preferably, L¹ is not further substituted (apart from the mandatorysubstituent L²-Z).

Preferably, the insulin moiety is attached to L¹ through the nitrogenN^(αA1) or through the nitrogen of a lysine side chain of the insulinmoiety.

Preferably, the insulin moiety is recombinant human insulin.

As shown in, e.g., formulae (Ia) to (Id) one hydrogen is replaced by thegroup L²-Z.

In general, L² can be attached to L¹ at any position apart from thereplacement of the hydrogen marked with an asterisk in formula (I).Preferably, one of the hydrogens given by R¹, R^(1a), R², R^(2a),R^(2b), R³, R^(3a), R⁴ directly or as hydrogen of the C₁₋₄ alkyl orfurther groups is replaced by L²-Z.

Furthermore, L¹ may be optionally further substituted. In general, anysubstituent may be used as far as the cleavage principle is notaffected. However it is preferred that L¹ is not further substituted.

Preferably, one or more further optional substituents are independentlyselected from the group consisting of halogen; CN; COOR⁹; OR⁹; C(O)R⁹;C(O)N(R⁹R^(9a)); S(O)₂N(R⁹R^(9a)); S(O)N(R⁹R^(9a)); S(O)₂R⁹; S(O)R⁹;N(R⁹)S(O)₂N(R^(9a)R^(9b)); SR⁹; N(R⁹R^(9a)); NO₂; OC(O)R⁹;N(R⁹)C(O)R^(9a); N(R⁹)S(O)₂R^(9a); N(R⁹)S(O)R^(9a); N(R⁹)C(O)OR^(9a);N(R⁹)C(O)N(R^(9a)R^(9b)); OC(O)N(R⁹R^(9a)); T; C₁₋₅₀ alkyl; C₂₋₅₀alkenyl; or C₂₋₅₀ alkynyl, wherein T; C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; andC₂₋₅₀ alkynyl are optionally substituted with one or more R¹⁰, which arethe same or different and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of T, —C(O)O—; —O—; —C(O)—; —C(O)N(R¹¹)—;—S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—; —N(R¹¹)S(O)₂N(R^(11a))—;—N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—; —N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—;—N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and —OC(O)N(R¹¹R^(11a));

R⁹, R^(9a), R^(9b) are independently selected from the group consistingof H; T; and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; or C₂₋₅₀ alkynyl, wherein T;C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally substitutedwith one or more R¹⁰, which are the same or different and wherein C₁₋₅₀alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally interrupted byone or more groups selected from the group consisting of T, —C(O)O—;—O—; —C(O)—; —C(O)N(R¹¹)—; —S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—;—N(R¹¹)S(O)₂N(R^(11a))—; —S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—;—N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—; —N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and—OC(O)N(R¹¹R^(11a));

T is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered heterocyclyl; or9 to 11 membered heterobicyclyl, wherein T is optionally substitutedwith one or more R¹⁰, which are the same or different;

R¹⁰ is halogen; CN; oxo (═O); COOR¹²; OR¹²; C(O)R¹²; C(O)N(R¹²R^(12a));S(O)₂N(R¹²R^(12a)); S(O)N(R¹²R^(12a)); S(O)₂R¹²; S(O)R¹²;N(R¹²)S(O)₂N(R^(12a)R^(12b)); SR¹²; N(R¹²R^(12a)); NO₂; OC(O)R¹²;N(R¹²)C(O)R^(12a); N(R¹²)S(O)₂R^(12a); N(R¹²)S(O)R^(12a);N(R¹²)C(O)OR^(12a); N(R¹²)C(O)N(R^(12a)R^(12b)); OC(O)N(R¹²R^(12a)); orC₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionally substituted with one ormore halogen, which are the same or different;

R¹¹, R^(11a), R¹², R^(12a), R^(12b) are independently selected from thegroup consisting of H; or C₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different.

The term “interrupted” means that between two carbons a group isinserted or at the end of the carbon chain between the carbon andhydrogen.

L² is a single chemical bond or a spacer. In case L² is a spacer, it ispreferably defined as the one or more optional substituents definedabove, provided that L² is substituted with Z.

Accordingly, when L² is other than a single chemical bond, L²-Z isCOOR⁹; OR⁹; C(O)R⁹; C(O)N(R⁹R^(9a)); S(O)₂N(R⁹R^(9a)); S(O)N(R⁹R^(9a));S(O)₂R⁹; S(O)R⁹; N(R⁹)S(O)₂N(R^(9a)R^(9b)); SR⁹; N(R⁹R^(9a)); OC(O)R⁹;N(R⁹)C(O)R^(9a); N(R⁹)S(O)₂R^(9a); N(R⁹)S(O)R^(9a); N(R⁹)C(O)OR^(9a);N(R⁹)C(O)N(R^(9a)R^(9b)); OC(O)N(R⁹R^(9a)); T; C₁₋₅₀ alkyl; C₂₋₅₀alkenyl; or C₂₋₅₀ alkynyl, wherein T; C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; andC₂₋₅₀ alkynyl are optionally substituted with one or more R¹⁰, which arethe same or different and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of -T-, —C(O)O—; —O—; —C(O)—; —C(O)N(R¹¹)—;—S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—; —N(R¹¹)S(O)₂N(R^(11a))—;—S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—; —N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—;—N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and —OC(O)N(R¹¹R^(11a));

R⁹, R^(9a), R^(9b) are independently selected from the group consistingof H; Z; T; and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; or C₂₋₅₀ alkynyl, wherein T;C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally substitutedwith one or more R¹⁰, which are the same or different and wherein C₁₋₅₀alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally interrupted byone or more groups selected from the group consisting of T, —C(O)O—;—O—; —C(O)—; —C(O)N(R¹¹)—; —S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—;—N(R¹¹)S(O)₂N(R^(11a))—; —S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—;—N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—; —N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and—OC(O)N(R¹¹R^(11a));

T is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered heterocyclyl; or9 to 11 membered heterobicyclyl, wherein t is optionally substitutedwith one or more R¹⁰, which are the same or different;

R¹⁰ is Z; halogen; CN; oxo (═O); COOR¹²; OR¹²; C(O)R¹²;C(O)N(R¹²R^(12a)); S(O)₂N(R¹²R^(12a)); S(O)N(R¹²R^(12a)); S(O)₂R¹²;S(O)R¹²; N(R¹²)S(O)₂N(R^(12a)R^(12b)); SR¹²; N(R¹²R^(12a)); NO₂;OC(O)R¹²; N(R¹²)C(O)R^(12a); N(R¹²)S(O)₂R^(12a); N(R¹²)S(O)R^(12a);N(R¹²)C(O)OR^(12a); N(R¹²)C(O)N(R^(12a)R^(12b)); OC(O)N(R¹²R^(12a)); orC₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionally substituted with one ormore halogen, which are the same or different;

R¹¹, R^(11a), R¹², R^(12a), R^(12b) are independently selected from thegroup consisting of H; Z; or C₁₋₆ alkyl, wherein C₁₋₆ alkyl isoptionally substituted with one or more halogen, which are the same ordifferent;

provided that only one of R⁹, R^(9a), R^(9b), R¹⁰, R¹¹, R^(11a), R¹²,R^(12a), R^(12b) is Z.

More preferably, L² is a C₁₋₂₀ alkyl chain, which is optionallyinterrupted by one or more groups independently selected from —O—; andC(O)N(R^(3aa)); optionally substituted with one or more groupsindependently selected from OH; and C(O)N(R^(3aa)R^(3aaa)); and whereinR^(3aa), R^(3aaa) are independently selected from the group consistingof H; and C₁₋₄ alkyl.

Preferably, L² has a molecular weight in the range of from 14 g/mol to750 g/mol.

Preferably, L² is attached to Z via a terminal group selected from

In case L² has such terminal group it is furthermore preferred that L²has a molecular weight in the range of from 14 g/mol to 500 g/molcalculated without such terminal group.

Preferably, the covalent attachment formed between the linker andhydrogel Z is a permanent bond.

Preferably, the hydrogel Z is a biodegradable polyethylene glycol (PEG)based water-insoluble hydrogel. The term “PEG based” as understoodherein means that the mass proportion of PEG chains in the hydrogel isat least 10% by weight, preferably at least 25%, based on the totalweight of the hydrogel. The remainder can be made up of other spacersand/or oligomers or polymers, such as oligo- or polylysines.

Moreover the term “water-insoluble” refers to a swellablethree-dimensionally crosslinked molecular network forming the hydrogel.The hydrogel if suspended in a large surplus of water or aqueous bufferof physiological osmolality may take up a substantial amount of water,e.g. up to 10-fold on a weight per weight basis, and is thereforeswellable but after removing excess water still retains the physicalstability of a gel and a shape. Such shape may be of any geometry and itis understood that such an individual hydrogel object is to beconsidered as a single molecule consisting of components wherein eachcomponent is connected to each other component through chemical bonds.

According to this invention, the hydrogel may be composed of backbonemoieties interconnected by hydrolytically degradable bonds.

Preferably, L² is connected to a backbone moiety

Preferably, the backbone moiety has a molecular weight in the range offrom 1 kDa to 20 kDa, more preferably from 1 kDa to 15 kDa and even morepreferably from 1 kDa to 10 kDa. The backbone moieties are preferablyalso PEG-based comprising one or more PEG chains.

In a hydrogel carrying drug-linker conjugates according to theinvention, a backbone moiety is characterized by a number of functionalgroups, comprising interconnected biodegradable functional groups andhydrogel-connected drug-linker conjugates, and optionally cappinggroups. This means that a backbone moiety is characterized by a numberof hydrogel-connected drug-linker conjugates; functional groups,comprising biodegradable interconnected functional groups; andoptionally capping groups. Preferably, the sum of interconnectedbiodegradable functional groups and drug-linker conjugates and cappinggroups is 16-128, preferred 20-100, more preferred 24-80 and mostpreferred 30-60.

Preferably, the sum of interconnected functional groups andhydrogel-connected drug-linker conjugates and capping groups of abackbone moiety is equally divided by the number of PEG-based polymericchains extending from the branching core. For instance, if there are 32interconnected functional groups and hydrogel-connected drug-linkerconjugates and capping groups, eight groups may be provided by each ofthe four PEG-based polymeric chains extending from the core, preferablyby means of dendritic moieties attached to the terminus of eachPEG-based polymeric chain. Alternatively, four groups may be provided byeach of eight PEG-based polymeric chains extending from the core or twogroups by each of sixteen PEG-based polymeric chains. If the number ofPEG-based polymeric chains extending from the branching core does notallow for an equal distribution, it is preferred that the deviation fromthe mean number of the sum of interconnected functional groups andhydrogel-connected drug-linker conjugates and capping groups perPEG-based polymeric chain is kept to a minimum.

In such carrier-linked prodrugs according to the invention, it isdesirable that almost all drug release (>90%) has occurred before asignificant amount of release of the backbone moieties (<10%) has takenplace. This can be achieved by adjusting the carrier-linked prodrug'shalf-life versus the degradation kinetics of the hydrogel according tothe invention.

Preferentially, a backbone moiety is characterized by having a branchingcore, from which at least three PEG-based polymeric chains extend.Accordingly, in a preferred aspect of the present invention the backbonereagent comprises a branching core, from which at least three PEG-basedpolymeric chains extend. Such branching cores may be comprised of poly-or oligoalcohols in bound form, preferably pentaerythritol,tripentaerythritol, hexaglycerine, sucrose, sorbitol, fructose,mannitol, glucose, cellulose, amyloses, starches, hydroxyalkyl starches,polyvinylalcohols, dextranes, hyualuronans, or branching cores may becomprised of poly- or oligoamines such as ornithine, diaminobutyricacid, trilysine, tetralysine, pentalysine, hexylysine, heptalysine,octalysine, nonalysine, decalysine, undecalysine, dodecalysine,tridecalysine, tetradecalysine, pentadecalysine or oligolysines,polyethyleneimines, polyvinylamines in bound form.

Preferably, the branching core extends three to sixteen PEG-basedpolymeric chains, more preferably four to eight. Preferred branchingcores may be comprised of pentaerythritol, ornithine, diaminobutyricacid, trilysine, tetralysine, pentalysine, hexylysine, heptalysine oroligolysine, low-molecular weight PEI, hexaglycerine, tripentaerythritolin bound form. Preferably, the branching core extends three to sixteenPEG-based polymeric chains, more preferably four to eight. Preferably, aPEG-based polymeric chain is a linear poly(ethylene glycol) chain, ofwhich one end is connected to the branching core and the other to ahyperbranched dendritic moiety. It is understood that a polymericPEG-based chain may be terminated or interrupted by alkyl or aryl groupsoptionally substituted with heteroatoms and chemical functional groups.

Preferably, a PEG-based polymeric chain is a suitably substitutedpolyethylene glycol derivative (PEG based).

Preferred structures for corresponding PEG-based polymeric chainsextending from a branching core contained in a backbone moiety aremulti-arm PEG derivatives as, for instance, detailed in the productslist of JenKem Technology, USA (accessed by download fromwww.jenkemusa.com on Jul. 28, 2009), 4ARM-PEG Derivatives(pentaerythritol core), 8ARM-PEG Derivatives (hexaglycerin core) and8ARM-PEG Derivatives (tripentaerythritol core). Most preferred are 4armPEG Amine (pentaerythritol core) and 4arm PEG Carboxyl (pentaerythritolcore), 8arm PEG Amine (hexaglycerin core), 8arm PEG Carboxyl(hexaglycerin core), 8arm PEG Amine (tripentaerythritol core) and 8armPEG Carboxyl (tripentaerythritol core). Preferred molecular weights forsuch multi-arm PEG-derivatives in a backbone moiety are 1 kDa to 20 kDa,more preferably 1 kDa to 15 kDa and even more preferably 1 kDa to 10kDa.

It is understood that the terminal amine groups of the above mentionedmulti-arm molecules are present in bound form in the backbone moiety toprovide further interconnected functional groups and reactive functionalgroups of a backbone moiety.

It is preferred that the sum of interconnected functional groups andreactive functional groups of a backbone moiety is equally divided bythe number of PEG-based polymeric chains extending from the branchingcore. If the number of PEG-based polymeric chains extending from thebranching core does not allow for an equal distribution, it is preferredthat the deviation from the mean number of the sum of interconnected andreactive functional groups per PEG-based polymeric chain is kept to aminimum.

More preferably, the sum of interconnected and reactive functionalgroups of a backbone moiety is equally divided by the number ofPEG-based polymeric chains extending from the branching core. Forinstance, if there are 32 interconnected functional groups and reactivefunctional groups, eight groups may be provided by each of the fourPEG-based polymeric chains extending from the core, preferably by meansof dendritic moieties attached to the terminus of each PEG-basedpolymeric chain. Alternatively, four groups may be provided by each ofeight PEG-based polymeric chains extending from the core or two groupsby each of sixteen PEG-based polymeric chains.

Such additional functional groups may be provided by dendritic moieties.Preferably, each dendritic moiety has a molecular weight in the range offrom 0.4 kDa to 4 kDa, more preferably 0.4 kDa to 2 kDa. Preferably,each dendritic moiety has at least 3 branchings and at least 4 reactivefunctional groups, and at most 63 branchings and 64 reactive functionalgroups, preferred at least 7 branchings and at least 8 reactivefunctional groups and at most 31 branchings and 32 reactive functionalgroups.

Examples for such dendritic moieties are comprised of trilysine,tetralysine, pentalysine, hexylysine, heptalysine, octalysine,nonalysine, decalysine, undecalysine, dodecalysine, tridecalysine,tetradecalysine, pentadecalysine, hexadecalysine, heptadecalysine,octadecalysine, nonadecalysine in bound form. Examples for suchpreferred dendritic moieties are comprised of trilysine, tetralysine,pentalysine, hexylysine, heptalysine in bound form, most preferredtrilysine, pentalysine or heptalysine, ornithine, diaminobutyric acid inbound form.

Most preferably, the hydrogel carrier of the present invention ischaracterized in that the backbone moiety has a quarternary carbon offormula C(A-Hyp)₄, wherein each A is independently a poly(ethyleneglycol) based polymeric chain terminally attached to the quarternarycarbon by a permanent covalent bond and the distal end of the PEG-basedpolymeric chain is covalently bound to a dendritic moiety Hyp, eachdendritic moiety Hyp having at least four functional groups representingthe interconnected functional groups and reactive functional groups.

Preferably, each A is independently selected from the formula—(CH₂)_(n1)(OCH₂CH₂)_(n)X—, wherein n1 is 1 or 2; n is an integer in therange of from 5 to 50; and X is a chemical functional group covalentlylinking A and Hyp.

Preferably, A and Hyp are covalently linked by an amide linkage.

Preferably, the dendritic moiety Hyp is a hyperbranched polypeptide.Preferably, the hyperbranched polypeptide comprises lysine in boundform. Preferably, each dendritic moiety Hyp has a molecular weight inthe range of from 0.4 kDa to 4 kDa. It is understood that a backbonemoiety C(A-Hyp)₄ can consist of the same or different dendritic moietiesHyp and that each Hyp can be chosen independently. Each moiety Hypconsists of between 5 and 32 lysines, preferably of at least 7 lysines,i.e. each moiety Hyp is comprised of between 5 and 32 lysines in boundform, preferably of at least 7 lysines in bound form. Most preferablyHyp is comprised of heptalysinyl.

The reaction of polymerizable functional groups a backbone reagent, morespecifically of Hyp with the polymerizable functional groups ofpolyethyleneglycol based crosslinker reagents results in a permanentamide bond.

Preferably, C(A-Hyp)₄ has a molecular weight in the range of from 1 kDato 20 kDa, more preferably 1 kDa to 15 kDa and even more preferably 1kDa to 10 kDa.

One preferred backbone moiety is shown below, dashed lines indicateinterconnecting biodegradable linkages to crosslinker moieties and n isan integer of from 5 to 50:

Biodegradability of the hydrogels according to the present invention isachieved by introduction of hydrolytically degradable bonds.

The terms “hydrolytically degradable”, “biodegradable” or“hydrolytically cleavable”, “auto-cleavable”, or “self-cleavage”,“self-cleavable”, “transient” or “temporary” refers within the contextof the present invention to bonds and linkages which arenon-enzymatically hydrolytically degradable or cleavable underphysiological conditions (aqueous buffer at pH 7.4, 37° C.) withhalf-lives ranging from one hour to three months, including, but are notlimited to, aconityls, acetals, amides, carboxylic anhydrides, esters,imines, hydrazones, maleamic acid amides, ortho esters, phosphamides,phosphoesters, phosphosilyl esters, silyl esters, sulfonic esters,aromatic carbamates, combinations thereof, and the like.

If present in a hydrogel according to the invention as degradableinterconnected functional group, preferred biodegradable linkages arecarboxylic esters, carbonates, phosphoesters and sulfonic acid estersand most preferred are carboxylic esters or carbonates.

Permanent linkages are non-enzymatically hydrolytically degradable underphysiological conditions (aqueous buffer at pH 7.4, 37° C.) withhalf-lives of six months or longer, such as, for example, amides.

To introduce the hydrolytically cleavable bonds into the hydrogelcarrier of the invention, the backbone moieties can be directly linkedto each other by means of biodegradable bonds.

In one embodiment, the backbone moieties of the biodegradable hydrogelcarrier may be linked together directly, i.e. without crosslinkermoieties. The hyperbranched dendritic moieties of two backbone moietiesof such biodegradable hydrogel may either be directly linked through aninterconnected functional group that connects the two hyperbrancheddendritic moieties. Alternatively, two hyperbranched dendritic moietiesof two different backbone moieties may be interconnected through twospacer moieties connected to a backbone moiety and on the other sideconnected to a crosslinking moiety separated by an interconnectedfunctional groups.

Alternatively, backbone moieties may be linked together throughcrosslinker moieties, each crosslinker moiety is terminated by at leasttwo of the hydrolytically degradable bonds. In addition to theterminating degradable bonds, the crosslinker moieties may containfurther biodegradable bonds. Thus, each end of the crosslinker moietylinked to a backbone moiety comprises a hydrolytically degradable bond,and additional biodegradable bonds may optionally be present in thecrosslinker moiety.

Preferably, the biodegradable hydrogel carrier is composed of backbonemoieties interconnected by hydrolytically degradable bonds and thebackbone moieties are linked together through crosslinker moieties.

The biodegradable hydrogel carrier may contain one or more differenttypes of crosslinker moieties, preferably one. The crosslinker moietymay be a linear or branched molecule and preferably is a linearmolecule. In a preferred embodiment of the invention, the crosslinkermoiety is connected to backbone moieties by at least two biodegradablebonds.

Preferably, crosslinker moieties have a molecular weight in the range offrom 60 Da to 5 kDa, more preferably, from 0.5 kDa to 4 kDa, even morepreferably from 1 kDa to 4 kDa, even more preferably from 1 kDa to 3kDa. In one embodiment, a crosslinker moiety consists of a polymer.

In addition to oligomeric or polymeric crosslinking moieties,low-molecular weight crosslinking moieties may be used, especially whenhydrophilic high-molecular weight backbone moieties are used for theformation of a biodegradable hydrogel according to the invention.

Preferably, the poly(ethylene glycol) based crosslinker moieties arehydrocarbon chains comprising ethylene glycol units, optionallycomprising further chemical functional groups, wherein the poly(ethyleneglycol) based crosslinker moieties comprise at least each methyleneglycol units, wherein m is an integer in the range of from 3 to 100,preferably from 10 to 70. Preferably, the poly(ethylene glycol) basedcrosslinker moieties have a molecular weight in the range of from 0.5kDa to 5 kDa.

If used in reference to a crosslinker moiety or a PEG-based polymericchain connected to a branching core, the term “PEG-based” refers to acrosslinker moiety or PEG-based polymeric chain comprising at least 20weight % ethylene glycol moieties.

In one embodiment, monomers constituting the polymeric crosslinkermoieties are connected by biodegradable bonds. Such polymericcrosslinker moieties may contain up to 100 biodegradable bonds or more,depending on the molecular weight of the crosslinker moiety and themolecular weight of the monomer units. Examples for such crosslinkermoieties are poly(lactic acid) or poly(glycolic acid) based polymers. Itis understood that such poly(lactic acid) or poly(glycolic acid) chainmay be terminated or interrupted by alkyl or aryl groups and that theymay optionally be substituted with heteroatoms and chemical functionalgroups.

Preferably, the crosslinker moieties are PEG based, preferablyrepresented by only one PEG based molecular chain. Preferably, thepoly(ethylene glycol) based crosslinker moieties are hydrocarbon chainscomprising ethylene glycol units, optionally comprising further chemicalfunctional groups, wherein the poly(ethylene glycol) based crosslinkermoieties comprise at least each methylene glycol units, wherein m is aninteger in the range of from 3 to 100, preferably from 10 to 70.Preferably, the poly(ethylene glycol) based crosslinker moieties have amolecular weight in the range of from 0.5 kDa to 5 kDa.

In a preferred embodiment of the present invention the crosslinkermoiety consists of PEG, which is symmetrically connected through esterbonds to two alpha, omega-aliphatic dicarboxylic spacers provided bybackbone moieties connected to the hyperbranched dendritic moietythrough permanent amide bonds.

The dicarboxylic acids of the spacer moieties connected to a backbonemoiety and on the other side is connected to a crosslinking moietyconsist of 3 to 12 carbon atoms, most preferably between 5 and 8 carbonatoms and may be substituted at one or more carbon atom. Preferredsubstituents are alkyl groups, hydroxyl groups or amido groups orsubstituted amino groups. One or more of the aliphatic dicarboxylicacid's methylene groups may optionally be substituted by O or NH oralkyl-substituted N. Preferred alkyl is linear or branched alkyl with 1to 6 carbon atoms.

Preferably, there is a permanent amide bond between the hyperbrancheddendritic moiety and the spacer moiety connected to a backbone moietyand on the other side is connected to a crosslinking moiety.

One preferred crosslinker moiety is shown below; dashed lines indicateinterconnecting biodegradable linkages to backbone moieties:

wherein n is an integer of from 5 to 50.

Preferably, the hydrogel carrier is composed of backbone moietiesinterconnected by hydrolytically degradable bonds.

More preferably, the backbone moieties comprise a branching core of thefollowing formula:

-   -   wherein the dashed line indicates attachment to the remainder of        the backbone moiety.

More preferably, the backbone moieties comprise a structure of thefollowing formula:

-   -   wherein n is an integer of from 5 to 50 and the dashed line        indicates attachment to the remainder of the backbone moiety.

Preferably, backbone moiety comprises a hyperbranched moiety Hyp.

More preferably, the backbone moiety comprises a hyperbranched moietyHyp of the following formula:

-   -   wherein the dashed lines indicate attachment to the rest of the        molecule and carbon atoms marked with asterisks indicate        S-configuration.

Preferably, the backbone moieties are attached to at least one spacer ofthe following formula:

-   -   wherein one of the dashed lines indicates attachment to the        hyperbranched moiety Hyp and the second dashed line indicates        attachment to the rest of the molecule; and    -   wherein m is an integer of from 2 to 4.

Preferably, the backbone moieties are linked together throughcrosslinker moieties having the following structure

wherein

q is an integer from 3 to 100, preferably from 5 to 50.

In hydrogel prodrugs of the invention, the hydrolysis rate of thebiodegradable bonds between backbone moieties and crosslinker moietiesis influenced or determined by the number and type of connected atomsadjacent to the PEG-ester carboxy group. For instance, by selecting fromsuccinic, adipic or glutaric acid for PEG ester formation it is possibleto vary the degradation half-lives of the biodegradable hydrogel carrieraccording to the invention.

Preferably, L² is attached to Z through a thiosuccinimide group which inturn is attached to the hydrogel's backbone moiety through a spacer,such as an oligoethylene glycol chain. Preferably, the linkage of thisspacer chain to the backbone moiety is a permanent bond, preferably anamide bond.

Biodegradability of the hydrogels according to the present invention isachieved, by introduction of hydrolytically degradable bonds.

For interconnected functional groups, the term “hydrolyticallydegradable” refers within the context of the present invention tolinkages which are non-enzymatically hydrolytically degradable underphysiological conditions (aqueous buffer at pH 7.4, 37° C.) withhalf-lives ranging from one hour to three months, include, but are notlimited to, aconityls, acetals, carboxylic anhydrides, esters, imines,hydrazones, maleamic acid amides, ortho esters, phosphamides,phosphoesters, phosphosilyl esters, silyl esters, sulfonic esters,aromatic carbamates, combinations thereof, and the like. Preferredbiodegradable linkages are carboxylic esters, carbonates, phosphoestersand sulfonic acid esters and most preferred are carboxylic esters orcarbonates. It is understood that for in vitro studies acceleratedconditions like, for example, pH 9, 37° C., aqueous buffer, may be usedfor practical purposes.

Permanent linkages are non-enzymatically hydrolytically degradable underphysiological conditions (aqueous buffer at pH 7.4, 37° C.) withhalf-lives of six months or longer, such as, for example, amides.

The degradation of the biodegradable hydrogel carrier according to theinvention is a multi-step reaction where a multitude of degradable bondsis cleaved resulting in degradation products which may be water-solubleor water-insoluble. However, water-insoluble degradation products mayfurther comprise degradable bonds so that they can be cleaved in thatwater-soluble degradation products are obtained. These water-solubledegradation products may comprise one or more backbone moieties. It isunderstood that released backbone moieties may, for instance, bepermanently conjugated to spacer or blocking or linker groups oraffinity groups and/or prodrug linker degradation products and that alsowater-soluble degradation products may comprise degradable bonds.

The structures of the branching core, PEG-based polymeric chains,hyperbranched dendritic moieties and moieties attached to thehyperbranched dendritic moieties can be inferred from the correspondingdescriptions provided in the sections covering the hydrogel carriers ofthe present invention. It is understood that the structure of adegradant depends on the type of hydrogel according to the inventionundergoing degradation.

The total amount of backbone moieties can be measured in solution aftercomplete degradation of the hydrogel according to the invention, andduring degradation, fractions of soluble backbone degradation productscan be separated from the insoluble hydrogel according to the inventionand can be quantified without interference from other solubledegradation products released from the hydrogel according to theinvention. A hydrogel object according to the invention may be separatedfrom excess water of buffer of physiological osmolality by sedimentationor centrifugation. Centrifugation may be performed in such way that thesupernatant provides for at least 10% of the volume of the swollenhydrogel according to the invention. Soluble hydrogel degradationproducts remain in the aqueous supernatant after such sedimentation orcentrifugation step, and water-soluble degradation products comprisingone or more backbone moieties are detectable by subjecting aliquots ofsuch supernatant to suitable separation and/or analytical methods.

Preferably, water-soluble degradation products may be separated fromwater-insoluble degradation products by filtration through 0.45 μmfilters, after which the water-soluble degradation products can be foundin the flow-through. Water-soluble degradation products may also beseparated from water-insoluble degradation products by a combination ofa centrifugation and a filtration step.

For instance the backbone moieties may carry groups that exhibit UVabsorption at wavelengths where other degradation products do notexhibit UV absorption. Such selectively UV-absorbing groups may bestructural components of the backbone moiety such as amide bonds or maybe introduced into the backbone by attachment to its reactive functionalgroups by means of aromatic ring systems such as indoyl groups.

In such hydrogel-linked insulin prodrugs according to the invention, itis desirable that almost all insulin release (>90%) has occurred beforea significant amount of release of the backbone degradation products(<10%) has taken place. This can be achieved by adjusting thehydrogel-linked insulin prodrug's half-life versus the hydrogeldegradation kinetics.

Preferably, are insulin prodrugs have the structure of formula (IIa) or(IIb)

-   -   wherein N^(ε)-Insulin refers to insulin connected via one lysine        side chain; or

Preferably, the hydrogel in (IIa) or (IIb) is a biodegradablepolyethylene glycol (PEG) based water-insoluble hydrogel.

Preferably, the hydrogel in (IIa) or (IIb) is composed of backbonemoieties interconnected by hydrolytically degradable bonds.

More preferably, the backbone moieties comprise a branching core of thefollowing formula:

-   -   wherein the dashed line indicates attachment to the remainder of        the backbone moiety.

More preferably, the backbone moieties comprise a structure of thefollowing formula:

-   -   wherein n is an integer of from 5 to 50 and the dashed line        indicates attachment to the rest of the molecule.

Preferably, backbone moiety comprises a hyperbranched moiety Hyp.

More preferably, the backbone moiety comprises a hyperbranched moietyHyp of the following formula:

-   -   wherein the dashed lines indicate attachment to the rest of the        molecule and carbon atoms marked with asterisks indicate        S-configuration.

Preferably, the backbone moieties are attached to at least one spacer ofthe following formula:

-   -   wherein one of the dashed lines indicates attachment to the        hyperbranched moiety Hyp and the second dashed line indicates        attachment to the rest of the molecule; and    -   wherein m is an integer of from 2 to 4.

Preferably, the backbone moieties are attached to at least one spacer ofthe following formula:

-   -   wherein the dashed line marked with the asterisk indicates the        bond between the hydrogel and the N of the thiosuccinimide        group,    -   wherein the other dashed line indicates attachment to Hyp, and    -   wherein p is an integer of from 0 to 10.

Preferably, the backbone moieties are linked together throughcrosslinker moieties having the following structure

-   -   wherein    -   q is an integer from 3 to 100;

The hydrolysis rate of the biodegradable bonds between backbone andcrosslinker moieties is determined by the number and type of connectedatoms adjacent to the PEG-ester carboxy group. For instance by selectingfrom succinic, adipic or glutaric acid for PEG ester formation it ispossible to vary the degradation half-lives of the crosslinker.

The hydrogel-linked insulin prodrug of the present invention can beprepared starting from the hydrogel of the present invention byconvenient methods known in the art. It is clear to a practitioner inthe art that several routes exist. For example the prodrug linkermentioned above to which the biologically active moiety is covalentlyattached can be reacted with the reactive functional groups of thehydrogel of the present invention with or with already bearing theactive moiety in part or as whole.

In a preferable method of preparation, the hydrogel is generated throughchemical ligation reactions. The hydrogel may be formed from twomacromolecular educts with complementary functionalities which undergo areaction such as a condensation or addition. One of these startingmaterials is a crosslinker reagent with at least two identicalfunctional groups and the other starting material is ahomomultifunctional backbone reagent. Suitable functional groups presenton the crosslinker reagent include terminal amino, carboxylic acid andderivatives, maleimide and other alpha,beta unsaturated Michaelacceptors like vinylsulfone, thiol, hydroxyl groups. Suitable functionalgroups present in the backbone reagent include but are not limited toamino, carboxylic acid and derivatives, maleimide and other alpha,betaunsaturated Michael acceptors like vinylsulfone, thiol, hydroxyl groups.

If the crosslinker reagent reactive functional groups are usedsubstoichiometrically with respect to backbone reactive functionalgroups, the resulting hydrogel will be a reactive hydrogel with freereactive functional groups attached to the backbone structure.

Optionally, the prodrug linker may be first conjugated to insulin andthe resulting insulin-prodrug linker conjugate may then react with thehydrogel's reactive functional groups. Alternatively, after activationof one of the functional groups of the prodrug linker, thelinker-hydrogel conjugate may be contacted with insulin in the secondreaction step and excess insulin may be removed by filtration afterconjugation of the insulin to the hydrogel-bound prodrug linker.

A preferred process for the preparation of a prodrug according to thepresent invention is as follows:

A preferred starting material for the backbone reagent synthesis is a4-arm PEG tetra amine or 8-arm PEG octa amine, with the PEG reagenthaving a molecular weight ranging from 2000 to 10000 Dalton, mostpreferably from 2000 to 5000 Da. To such multi-arm PEG-derivatives,lysine residues are coupled sequentially to form the hyperbranchedbackbone reagent. It is understood that the lysines can be partially orfully protected by protective groups during the coupling steps and thatalso the final backbone reagent may contain protective groups. Apreferred building block is bis-boc lysine. Alternatively, instead ofsequential additions of lysine residues, a dendritic poly-lysine moietymay be assembled first and subsequently coupled to the 4-arm PEG tetraamine or 8-arm PEG octa amine. It is desirable to obtain backbonereagent carrying 32 amino groups, consequently seven lysines would beattached to each arm of a 4-arm PEG, or five lysines would be attachedto each arm of a 8-arm PEG. In another embodiment, the multi-arm PEGderivative is a tetra- or octa carboxy PEG. In this case, the dendriticmoieties may be generated from glutaric or aspartic acid, and theresulting backbone reagent would carry 32 carboxy groups. It isunderstood that all or a fraction of the backbone reagent's functionalgroups may be present in a free form, as salts or conjugated toprotecting groups. It is understood that due to practical reasons thebackbone reagent's number of lysines per PEG-arm will be between six andseven, more preferably approximately seven.

A preferred backbone reagent is shown below:

Synthesis of the crosslinker reagent starts from a linear PEG chain witha molecular weight ranging from 0.2 to 5 kDa, more preferably from 0.6to 2 kDa, which is esterified with a half ester of a dicarboxylic acid,most adipic acid or glutaric acid. Preferred protecting group for halfester formation is the benzylic group. The resulting bis dicarboxylicacid PEG half esters are converted into more reactive carboxy compoundssuch as acyl chlorides or active esters, eg pentafluorophenyl orN-hydroxysuccinimide esters, most preferred N-hydroxysuccinimde esters,of which preferred selected structure is shown below.

Alternatively, the bis dicarboxylic acid PEG half esters may beactivated in the presence of a coupling agent such as DCC or HOBt orPyBOP.

In an alternative embodiment the backbone reagent carries carboxy groupsand the corresponding crosslinker reagent would be selected fromester-containing amino-terminated PEG-chains.

Backbone reagent and crosslinker reagent may be polymerized to form thehydrogel according to the invention using inverse emulsionpolymerization. After selecting the desired stoichiometry betweenbackbone and crosslinker functional groups, backbone and crosslinker aredissolved in DMSO and a suitable emulgator with an appropriatelyselected HLB value, preferably Arlacel P135, is employed to form aninverse emulsion using a mechanical stirrer and controlling the stirringspeed. Polymerization is initiated by the addition of a suitable base,preferably by N,N,N′,N′-tetramethylethylene diamine. After stirring foran appropriate amount of time, the reaction is quenched by the additionof an acid, such as acetic acid and water. The beads are harvested,washed, and fractionated according to particle size by mechanicalsieving. Optionally, protecting groups may be removed at this stage.

Further, such hydrogel according to the invention may be functionalizedwith a spacer carrying a different reactive functional group thanprovided by the hydrogel. For instance maleimide reactive functionalgroups may be introduced into the hydrogel by coupling a suitableheterobifunctional spacer such as Mal-PEG6-NHS to the hydrogel. Suchfunctionalized hydrogel can be further conjugated to insulin-linkerreagents, carrying a reactive thiol group on the linker moiety to formhydrogel-linked insulin prodrugs according to the present invention.

After loading the insulin-linker conjugate to the functionalizedmaleimido group-containing hydrogel, all remaining functional groups arecapped with a suitable blocking reagent, such as mercaptoethanol, toprevent undesired side-reactions.

In a preferred embodiment of the invention, an insulin-linker conjugatecarrying a free thiol group connected to the linker moiety, is reactedwith a maleimide-functionalized hydrogel at temperatures between roomtemperature and 4° C., more preferred at room temperature, in a bufferedaqueous solution of pH 2-5, preferably pH 2.5-4.5, more preferably pH3.0-4.0. Subsequently, the corresponding resultinginsulin-linker-hydrogel conjugate is treated with mercaptoethanol attemperatures between room temperature and 4° C., more preferred at roomtemperature, in a buffered aqueous solution of pH 2-5, preferably pH2.5-4.0, more preferably pH 2.5-3.5.

In another preferred embodiment of the invention, an insulin-linkerconjugate carrying a maleimide group connected to the linker moiety, isreacted with a thiol-functionalized hydrogel at temperatures betweenroom temperature and 4° C., more preferred at room temperature, in abuffered aqueous solution of pH 2-5, preferably pH 2.5-4.5, morepreferably pH 3.0-4.0. Subsequently, the corresponding resultinginsulin-linker-hydrogel conjugate is treated with a low molecular weightcompound comprising a maleimide group, preferably a maleimide-containingcompound of 100 to 300 Da, e.g. N-ethyl-maleimide, at temperaturesbetween room temperature and 4° C., more preferred at room temperature,in a buffered aqueous solution of pH 2-5, preferably pH 2.5-4.0, morepreferably pH 2.5-3.5.

Another aspect of the present invention is a process comprising thesteps of

-   -   (a) contacting an aqueous suspension comprising        maleimide-functionalized hydrogel microparticles with a solution        comprising an insulin-linker reagent carrying thiol groups at        temperatures between room temperature and 4° C. in a buffered        aqueous solution of pH 2-5, resulting in an        insulin-linker-hydrogel conjugate;    -   (b) optionally, treating the insulin-linker-hydrogel conjugate        from step (a) with a thiol-containing compound of 34 Da to 500        Da at temperatures between room temperature and 4° C. in a        buffered aqueous solution of pH 2-5.

Another aspect of the present invention is a process comprising thesteps of

-   -   (a) contacting an aqueous suspension comprising        thiol-functionalized hydrogel microparticles with a solution        comprising an insulin-linker reagent carrying maleimide groups        at temperatures between room temperature and 4° C. in a buffered        aqueous solution of pH 2-5, resulting in an        insulin-linker-hydrogel conjugate;    -   (b) optionally, treating the insulin-linker-hydrogel conjugate        from step (a) with a maleimide-containing compound of 100 to 300        Da at temperatures between room temperature and 4° C. in a        buffered aqueous solution of pH 2-5.

A particularly preferred method for the preparation of a prodrug of thepresent invention comprises the steps of

-   -   (a) reacting a compound of formula C(A′-X¹)₄, wherein A′-X¹        represents A before its binding to Hyp or a precursor of Hyp and        X¹ is a suitable functional group, with a compound of formula        Hyp′-X², wherein Hyp′-X² represents Hyp before its binding to A        or a precursor of Hyp and X² is a suitable functional group to        react with X¹;    -   (b) optionally reacting the resulting compound from step (a) in        one or more further steps to yield a compound of formula        C(A-Hyp)₄ having at least four functional groups;    -   (c) reacting the at least four functional groups of the        resulting compound from step (b) with a polyethyleneglycol based        crosslinker precursor, wherein the active ester groups of the        crosslinker precursor are used in a sub-stoichiometric amount        compared to the total number of reactive functional groups of        C(A-Hyp)₄ to yield a hydrogel;    -   (d) reacting remaining un-reacted functional groups        (representing the reactive functional groups of the backbone        comprised in the hydrogel) in the hydrogel backbone of step (c)        with a covalent conjugate of biologically active moiety and        transient prodrug linker or first reacting the un-reacted        functional groups with the transient prodrug linker and        subsequently with the biologically active moiety;    -   (e) optionally capping remaining un-reacted functional groups to        yield a prodrug of the present invention.

Specifically, hydrogels for the insulin prodrugs of the presentinvention are synthesized as follows:

For bulk polymerization, backbone reagent and crosslinker reagent aremixed in a ratio amine/active ester of 2:1 to 1.05:1.

Both backbone reagent and crosslinker reagent are dissolved in DMSO togive a solution with a concentration of 5 to 50 g per 100 mL, preferably7.5 to 20 g per 100 ml and most preferably 10 to 20 g per 100 ml.

To effect polymerization, 2 to 10% (vol.) N,N,N′,N′-tertramethylethylenediamine (TMEDA) are added to the DMSO solution containing crosslinkerreagent and backbone reagent and the mixture is shaken for 1 to 20 secand left standing. The mixture solidifies within less than 1 min.

Such hydrogel according to the invention is preferably comminuted bymechanical processes such as stirring, crushing, cutting pressing, ormilling, and optionally sieving.

For emulsion polymerization, the reaction mixture is comprised of thedispersed phase and the continuous phase.

For the dispersed phase, backbone reagent and crosslinker reagent aremixed in a ratio amine/active ester of 2:1 to 1.05:1 and are dissolvedin DMSO to give a to give a solution with a concentration of 5 to 50 gper 100 mL, preferably 7.5 to 20 g per 100 ml and most preferably 10 to20 g per 100 ml.

The continuous phase is any solvent, that is not miscible with DMSO, notbasic, aprotic and shows a viscosity lower than 10 Pa*s. Preferably, thesolvent is not miscible with DMSO, not basic, aprotic, shows a viscositylower than 2 Pa*s and is non-toxic. More preferably, the solvent is asaturated linear or branched hydrocarbon with 5 to 10 carbon atoms. Mostpreferably, the solvent is n-heptane.

To form an emulsion of the dispersed phase in the continuous phase, anemulsifier is added to the continuous phase before adding the dispersedphase. The amount of emulsifier is 2 to 50 mg per mL dispersed phase,more preferably 5 to 20 mg per mL dispersed phase, most preferably 10 mgper mL dispersed phase.

The emulsifier has an HLB-value of 3 to 8. Preferably, the emulsifier isa triester of sorbitol and a fatty acid or an poly(hydroxyl fattyacid)-poly(ethylene glycol) conjugate. More preferably, the emulsifieris an poly(hydroxy-fatty acid)-polyethylene glycol conjugate, with alinear poly(ethylene glycol) of a molecular weight in the range of from0.5 kDa to 5 kDa and poly(hydroxy-fatty acid) units of a molecularweight in the range of from 0.5 kDa to 3 kDa on each end of the chain.Most preferably, the emulsifier is poly(ethylene glycol)dipolyhydroxystearate, Cithrol DPHS (Cithrol DPHS, former Arlacel P135, CrodaInternational Plc).

Droplets of the dispersed phase are generated by stirring with an axialflow impeller with a geometry similar to stirrers such as Isojet,Intermig, Propeller (EKATO Rühr- and Mischtechnik GmbH, Germany)), mostpreferably similar to Isojet with a diameter of 50 to 90% of the reactordiameter. Preferably, stirring is initiated before addition of thedispersed phase. Stirrer speed is set to 0.6 to 1.7 m/s. The dispersedphase is added at room temperature, and the concentration of thedisperse phase is 2% to 70%, preferably 5 to 50%, more preferably 10 to40%, and most preferably 20 to 35% of the total reaction volume. Themixture of dispersed phase, emulsifier and continuous phase is stirredfor 5 to 60 min before adding the base to the effect polymerization.

5 to 10 equivalents (referred to each amide bond to be formed) of a baseare added to the mixture of dispersed and continuous phase. The base isaprotic, non nucleophilic and soluble in the disperse phase. Preferably,the base is aprotic, non nucleophilic, well soluble in both dispersephase and DMSO. More preferably, the base is aprotic, non nucleophilic,well soluble in both disperse phase and DMSO, an amine base andnon-toxic. Most preferably, the base is N,N,N′,N′-tertramethylethylenediamine (TMEDA). Stirring in the presence of base is continued for 1 to16 h.

During stirring, droplets of dispersed phase are hardened to becomecrosslinked hydrogel beads according to the invention which can becollected and fractionation according to size is performed on avibrational continuous sieving machine with a 75 μm and a 32 μm deck togive hydrogel microparticles according to the invention.

Another aspect of the present invention are insulin-linker conjugates offormula (IIIa) and (IIIb)

-   -   wherein N^(ε)-Insulin refers to insulin connected via one lysine        side chain; and

Another aspect of the present invention are insulin-linker reagentsD-L*,

-   -   wherein    -   D represents an insulin moiety; and    -   L* is a non-biologically active linker reagent represented by        formula (IV),

-   -   wherein the dashed line indicates the attachment to one of the        amino groups of the insulin by forming an amide bond;    -   X is C(R³R^(3a)); or N(R³);    -   R^(1a)R^(3a) are independently selected from the group        consisting H, NH(R^(2b)), N(R^(2b))C(O)R⁴ and C₁₋₄ alkyl;    -   R¹, R², R^(2a), R^(2b), R³, R⁴ are independently selected from        the group consisting of H and C₁₋₄ alkyl,

wherein L* is substituted with one L²* and optionally furthersubstituted, provided that the hydrogen marked with the asterisk informula (IV) is not replaced by a substituent and wherein

-   -   L²* is a spacer connected to L* and comprising a chemical        functional group intended for conjugation to a reactive        biodegradable hydrogel;

Preferably, R² in formula (IV) is replaced by L²*.

Preferably, R¹ in formula (IV) is replaced by L²*.

Preferably, X in formula (IV) is N(R³).

More preferably, X in formula (IV) is C(R³R^(3a)) and R^(3a) isN(R^(2b))C(O)R⁴.

More preferably, X in formula (IV) is C(R³R^(3a)) and R^(3a) is replacedby L²*.

Even more preferably, X in formula (IV) is C(R³R^(3a)), R^(3a) isN(R^(2b))-L²*.

Preferably, L* in formula (IV) is not further substituted.

Preferably, L²* in formula (IV) comprises a thiol group.

Preferably, L²* in formula (IV) comprises a maleimide group.

The hydrogel for the prodrug of the present invention can be obtainedfrom the preparation methods in form of microparticles. In a preferredembodiment of the invention, the reactive hydrogel is a shaped articlesuch as a mesh or a stent. Most preferably, the hydrogel is formed intomicroparticulate beads which can be administered as subcutaneous orintramuscular injection by means of a standard syringe. Such soft beadsmay have a diameter of between 1 and 500 micrometer.

Preferably, the microparticles have a diameter of between 10 and 100micrometer if suspended in an isotonic aqueous formulation buffer, mostpreferably a diameter of between 20 and 100 micrometer, most preferablya diameter of between 25 and 80 micrometer.

Preferably, the microparticles can be administered by injection througha needle smaller than 0.6 mm inner diameter, preferably through a needlesmaller than 0.3 mm inner diameter, more preferably through a needlesmaller than 0.225 mm inner diameter, even more preferably through aneedle smaller than 0.175 mm inner diameter, and most preferably througha needle small than 0.16 mm inner diameter.

It is understood that the terms “can be administered by injection”,“injectable” or “injectability” refer to a combination of factors suchas a certain force applied to a plunger of a syringe containing thebiodegradable hydrogel according to the invention swollen in a liquid ata certain concentration (w/v) and at a certain temperature, a needle ofa given inner diameter connected to the outlet of such syringe, and thetime required to extrude a certain volume of the biodegradable hydrogelaccording to the invention from the syringe through the needle.

In order to provide for injectability, a volume of 1 mL of the insulinprodrugs according to the invention swollen in water to a concentrationof at least 5% (w/v) and contained in a syringe holding a plunger of adiameter of 4.7 mm can be extruded at room temperature within 10 secondsby applying a force of less than 50 Newton.

Preferably injectability is achieved for an insulin prodrug according tothe invention swollen in water to a concentration of ca. 10% (w/v).

Furthermore a one-step process is provided to selectively acylate singlefree ε-amino groups found in the B-chain of insulin and its analoga withan acylating agent containing a protected thiol or other functionalgroup. For recombinant human insulin, insulin glargine and insulineaspart the site of acylation is the ε-amino group of LysB29, in the caseof insulin Lispro the site of acylation is the ε-amino group of LysB28,and in the case of insulin glulisine the site of acylation is theε-amino group of LysB3. It is understood that this process is notlimited to the before-mentioned insulin and insulin analoga, but can beapplied to other insulin analoga as long as they contain ε-amino groupsand the person skilled in the art will be able to identify thecorresponding lysine residue suitable for acylation.

Thus another aspect of the present invention is a process for acylatingthe ε-amino group of insulin or an insulin analog, having one or morefree α-amino groups and the free ε-amino group with an acylating agentcontaining one or more protected functional groups, which comprisesreacting the insulin or insulin analog with a soluble acylating agentcontaining one or more protected functional groups at a pH of 8.0 tobelow 9.0 in a polar solvent. It is understood that only such protectivegroups are to be used that are stable in the before mentionedconditions.

The reaction is carried out by reacting an acylating agent, such as alinker reagent, which contains one or more protected functional groups,with the ε-amino group of the insulin or insulin analog under basicconditions with a pH ranging from about 8.00 to below 9.0, preferably,from 8.0 to 8.9, more preferably, from 8.3 to 8.7 in a polar solvent,such as aqueous mixtures of, for example, methanol, ethanol, propanol,isopropanol, DMSO, DMF, NMP, dimethylacetamid, acetonitrile.

Another aspect of the present invention is an insulin compoundcharacterized by having an acyl group linked to the ε-nitrogen ofinsulin or an insulin analog and wherein such acyl group has one or moreprotected functional groups.

Another aspect of the present invention is a pharmaceutical compositioncomprising a prodrug of the present invention or a pharmaceuticallyacceptable salt thereof together with a pharmaceutically acceptableexcipient. The pharmaceutical composition is further described in thefollowing paragraphs.

The composition of insulin-hydrogel prodrug may be provided as asuspension composition or as a dry composition. Preferably, thepharmaceutical composition of insulin-hydrogel prodrug is a drycomposition. Suitable methods of drying are, for example, spray-dryingand lyophilization (freeze-drying). Preferably, the pharmaceuticalcomposition of insulin-hydrogel prodrug is dried by lyophilization.

Preferably, the insulin hydrogel prodrug is sufficiently dosed in thecomposition to provide therapeutically effective amount of insulin forat least three days in one application. More preferably, one applicationof the insulin hydrogel prodrug is sufficient for one week.

The pharmaceutical composition of insulin-hydrogel prodrug according tothe present invention contains one or more excipients.

Excipients used in parenteral compositions may be categorized asbuffering agents, isotonicity modifiers, preservatives, stabilizers,anti-adsorption agents, oxidation protection agents,viscosifiers/viscosity enhancing agents, or other auxiliary agents. Insome cases, these ingredients may have dual or triple functions. Thecompositions of insulin-hydrogel prodrugs according to the presentinvention contain one or more than one excipient, selected from thegroups consisting of:

-   (i) Buffering agents: physiologically tolerated buffers to maintain    pH in a desired range, such as sodium phosphate, bicarbonate,    succinate, histidine, citrate and acetate, sulphate, nitrate,    chloride, pyruvate. Antacids such as Mg(OH)₂ or ZnCO₃ may be also    used. Buffering capacity may be adjusted to match the conditions    most sensitive to pH stability-   (ii) Isotonicity modifiers: to minimize pain that can result from    cell damage due to osmotic pressure differences at the injection    depot. Glycerin and sodium chloride are examples. Effective    concentrations can be determined by osmometry using an assumed    osmolality of 285-315 mOsmol/kg for serum-   (iii) Preservatives and/or antimicrobials: multidose parenteral    preparations require the addition of preservatives at a sufficient    concentration to minimize risk of patients becoming infected upon    injection and corresponding regulatory requirements have been    established. Typical preservatives include m-cresol, phenol,    methylparaben, ethylparaben, propylparaben, butylparaben,    chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosol,    sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and    benzalkonium chloride-   (iv) Stabilizers: Stabilisation is achieved by strengthening of the    protein-stabilising forces, by destabilisation of the denatured    stater, or by direct binding of excipients to the protein.    Stabilizers may be amino acids such as alanine, arginine, aspartic    acid, glycine, histidine, lysine, proline, sugars such as glucose,    sucrose, trehalose, polyols such as glycerol, mannitol, sorbitol,    salts such as potassium phosphate, sodium sulphate, chelating agents    such as EDTA, hexaphosphate, ligands such as divalent metal ions    (zinc, calcium, etc.), other salts or organic molecules such as    phenolic derivatives. In addition, oligomers or polymers such as    cyclodextrins, dextran, dendrimers, PEG or PVP or protamine or HSA    may be used-   (v) Anti-adsorption agents: Mainly ionic or non-ionic surfactants or    other proteins or soluble polymers are used to coat or adsorb    competitively to the inner surface of the composition's or    composition's container. E.g., poloxamer (Pluronic F-68), PEG    dodecyl ether (Brij 35), polysorbate 20 and 80, dextran,    polyethylene glycol, PEG-polyhistidine, BSA and HSA and gelatines.    Chosen concentration and type of excipient depends on the effect to    be avoided but typically a monolayer of surfactant is formed at the    interface just above the CMC value-   (vi) Lyo- and/or cryoprotectants: During freeze- or spray drying,    excipients may counteract the destabilising effects caused by    hydrogen bond breaking and water removal. For this purpose sugars    and polyols may be used but corresponding positive effects have also    been observed for surfactants, amino acids, non-aqueous solvents,    and other peptides. Trehalose is particularly efficient at reducing    moisture-induced aggregation and also improves thermal stability    potentially caused by exposure of protein hydrophobic groups to    water. Mannitol and sucrose may also be used, either as sole    lyo/cryoprotectant or in combination with each other where higher    ratios of mannitol:sucrose are known to enhance physical stability    of a lyophilized cake. Mannitol may also be combined with trehalose.    Trehalose may also be combined with sorbitol or sorbitol used as the    sole protectant. Starch or starch derivatives may also be used-   (vii) Oxidation protection agents: antioxidants such as ascorbic    acid, ectoine, methionine, glutathione, monothioglycerol, morin,    polyethylenimine (PEI), propyl gallate, vitamin E, chelating agents    such as citric acid, EDTA, hexa phosphate, thioglycolic acid-   (viii) Viscosifiers or viscosity enhancers: retard settling of the    particles in the vial and syringe and are used in order to    facilitate mixing and resuspension of the particles and to make the    suspension easier to inject (i.e., low force on the syringe    plunger). Suitable viscosifiers or viscosity enhancers are, for    example, carbomer viscosifiers like Carbopol 940, Carbopol Ultrez    10, cellulose derivatives like hydroxypropylmethylcellulose    (hypromellose, HPMC) or diethylaminoethyl cellulose (DEAE or    DEAE-C), colloidal magnesium silicate (Veegum) or sodium silicate,    hydroxyapatite gel, tricalcium phosphate gel, xanthans, carrageenans    like Satia gum UTC 30, aliphatic poly(hydroxy acids), such as    poly(D,L- or L-lactic acid) (PLA) and poly(glycolic acid) (PGA) and    their copolymers (PLGA), terpolymers of D,L-lactide, glycolide and    caprolactone, poloxamers, hydrophilic poly(oxyethylene) blocks and    hydrophobic poly(oxypropylene) blocks to make up a triblock of    poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) (e.g.)    Pluronic®, polyetherester copolymer, such as a polyethylene glycol    terephthalate/polybutylene terephthalate copolymer, sucrose acetate    isobutyrate (SAIB), dextran or derivatives thereof, combinations of    dextrans and PEG, polydimethylsiloxane, collagen, chitosan,    polyvinyl alcohol (PVA) and derivatives, polyalkylimides,    poly(acrylamide-co-diallyldimethyl ammonium (DADMA)),    polyvinylpyrrolidone (PVP), glycosaminoglycans (GAGs) such as    dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin,    heparan sulfate, hyaluronan, ABA triblock or AB block copolymers    composed of hydrophobic A-blocks, such as polylactide (PLA) or    poly(lactide-co-glycolide) (PLGA), and hydrophilic B-blocks, such as    polyethylene glycol (PEG) or polyvinyl pyrrolidone. Such block    copolymers as well as the abovementioned poloxamers may exhibit    reverse thermal gelation behavior (fluid state at room temperature    to facilitate administration and gel state above sol-gel transition    temperature at body temperature after injection).-   (ix) Spreading or diffusing agent: modifies the permeability of    connective tissue through the hydrolysis of components of the    extracellular matrix in the intrastitial space such as but not    limited to hyaluronic acid, a polysaccharide found in the    intercellular space of connective tissue. A spreading agent such as    but not limited to hyaluronidase temporarily decreases the viscosity    of the extracellular matrix and promotes diffusion of injected    drugs.-   (x) Other auxiliary agents: such as wetting agents, viscosity    modifiers, antibiotics, hyaluronidase. Acids and bases such as    hydrochloric acid and sodium hydroxide are auxiliary agents    necessary for pH adjustment during manufacture.

Preferably, the composition of insulin-hydrogel prodrug contains one ormore than one viscosifier and/or viscosity modifying agent.

The term “excipient” preferably refers to a diluent, adjuvant, orvehicle with which the therapeutic is administered. Such pharmaceuticalexcipient can be sterile liquids, such as water and oils, includingthose of petroleum, animal, vegetable or synthetic origin, including butnot limited to peanut oil, soybean oil, mineral oil, sesame oil and thelike. Water is a preferred excipient when the pharmaceutical compositionis administered orally. Saline and aqueous dextrose are preferredexcipients when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions are preferably employed as liquid excipients for injectablesolutions. Suitable pharmaceutical excipients include starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like. Thecomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. These compositions can takethe form of solutions, suspensions, emulsions, tablets, pills, capsules,powders, sustained-release formulations and the like. The compositioncan be formulated as a suppository, with traditional binders andexcipients such as triglycerides. Oral formulation can include standardexcipients such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Examples of suitable pharmaceutical excipients are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain a therapeutically effective amount of the therapeutic,preferably in purified form, together with a suitable amount ofexcipient so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

In a general embodiment a pharmaceutical composition of the presentinvention whether in dry form or as a suspension or in another form maybe provided as single or multiple dose composition.

In one embodiment of the present invention, the dry composition ofinsulin-hydrogel prodrug is provided as a single dose, meaning that thecontainer in which it is supplied contains one pharmaceutical dose.

Thus in another aspect of the present invention the composition isprovided as a single dose composition.

Preferably, the suspension composition is a multiple dose composition,meaning that it contains more than one therapeutic dose. Preferably, amultiple dose composition contains at least 2 doses. Such multiple dosecomposition of insulin-hydrogel can either be used for differentpatients in need thereof or is intended for use in one patient, whereinthe remaining doses are stored after the application of the first doseuntil needed.

In another aspect of the present invention the composition is comprisedin a container. Preferably the container is a dual-chamber syringe.Especially the dry composition according to the present invention isprovided in a first chamber of the dual-chamber syringe andreconstitution solution is provided in a second chamber of thedual-chamber syringe.

Prior to applying the dry composition of insulin-hydrogel prodrug to apatient in need thereof, the dry composition is reconstituted.Reconstitution can take place in the container in which the drycomposition of insulin-hydrogel prodrug is provided, such as in a vial,syringe, dual-chamber syringe, ampoule, and cartridge. Reconstitution isdone by adding a predefined amount of reconstitution solution to the drycomposition. Reconstitution solutions are sterile liquids, such as wateror buffer, which may contain further additives, such as preservativesand/or antimicrobials. If the insulin-hydrogel prodrug composition isprovided as single dose, the reconstitution solution may contain one ormore preservative and/or antimicrobial. Preferably, the reconstitutionsolution is sterile water. If the composition of insulin-hydrogelprodrug is a multiple dose composition, it is preferred that thereconstitution solution contains one or more preservative and/orantimicrobial, such as, for example, benzylalcohol and cresol.

An additional aspect of the present invention relates to the method ofadministration of a reconstituted insulin hydrogel prodrug composition.The insulin hydrogel prodrug composition can be administered by methodsof injection or infusion, including intradermal, subcutaneous,intramuscular, intravenous, intraosseous, and intra peritoneal.

A further aspect is a method of preparing a reconstituted compositioncomprising a therapeutically effective amount of an insulin hydrogelprodrug, and optionally one or more pharmaceutically acceptableexcipients, wherein the insulin is transiently linked to a hydrogel, themethod comprising the step of

-   -   contacting the composition of the present invention with a        reconstitution solution.

Another aspect is a reconstituted composition comprising atherapeutically effective amount of a insulin hydrogel prodrug, andoptionally one or more pharmaceutically acceptable excipients, whereinthe insulin is transiently linked to a hydrogel obtainable by the methodabove.

Another aspect of the present invention is the method of manufacturing adry composition of insulin-hydrogel prodrug. In one embodiment, suchsuspension composition is made by

-   -   (i) admixing the insulin-hydrogel prodrug with one or more        excipients,    -   (ii) transferring amounts equivalent to single or multiple doses        into a suitable container,    -   (iii) drying the composition in said container, and    -   (iv) sealing the container.

Suitable containers are vials, syringes, dual-chamber syringes,ampoules, and cartridges.

Another aspect is a kit of parts. When the administration device issimply a hypodermic syringe then the kit may comprise the syringe, aneedle and a container comprising the dry insulin-hydrogel prodrugcomposition for use with the syringe and a second container comprisingthe reconstitution solution. In more preferred embodiments, theinjection device is other than a simple hypodermic syringe and so theseparate container with reconstituted insulin-hydrogel prodrug isadapted to engage with the injection device such that in use the liquidcomposition in the container is in fluid connection with the outlet ofthe injection device. Examples of administration devices include but arenot limited to hypodermic syringes and pen injector devices.Particularly preferred injection devices are the pen injectors in whichcase the container is a cartridge, preferably a disposable cartridge.

A preferred kit of parts comprises a needle and a container containingthe composition according to the present invention and optionallyfurther containing a reconstitution solution, the container beingadapted for use with the needle. Preferably, the container is adual-chamber syringe.

In another aspect, the invention provides a cartridge containing acomposition of insulin-hydrogel prodrug as hereinbefore described foruse with a pen injector device. The cartridge may contain a single doseor multiplicity of doses of insulin.

In one embodiment of the present invention the suspension composition ofinsulin-hydrogel prodrug does not only comprise an insulin-hydrogelprodrug and one or more than one excipients, but also other biologicallyactive agents, either in their free form or as prodrugs. Preferably,such additional one or more biologically active agent is a prodrug, morepreferably a hydrogel prodrug. Such biologically active agents include,but are not limited to, compounds of the following classes:

-   -   (i) Sulfonylureas, such as, for example, chlorpropamide,        tolazamide, tolbutamide, glyburide, glipizide, glimepiride, and        the like,    -   (ii) Meglitinides, such as, for example, repaglinide,    -   (iii) Glucagon-like Peptide-1 (GLP-1) and it's mimetics,        Glucose-insulinotropic peptide (GIP) and it's mimetics, Exendin        and it's mimetics, and Dipeptyl Protease Inhibitors (DPPIV),    -   (iv) Biguanides, such as, for example, metformin,    -   (v) Thiazolidinediones, such as, for example, rosiglitazone,        pioglitazone, troglitazone, isaglitazone (known as MCC-555),        2-[2-[(2R)-4-hexyl-3,4-dihydro-3-oxo-2H-1,4-benzoxazin-2-yl]ethoxy]-benzene        acetic acid, and the like    -   (vi) GW2570, and the like,    -   (vii) Retinoid-X receptor (RXR) modulators, such as, for        example, targretin, 9-cis-retinoic acid, and the like,    -   (viii) Other insulin sensitizing agents, such as, for example,        INS-1, PTP-1B inhibitors, GSK3 inhibitors, glycogen        phosphorylase a inhibitors, fructose-1,6-bisphosphatase        inhibitors, and the like,    -   (ix) Insulins, including regular or short-acting,        intermediate-acting, and long-acting insulins, inhaled insulin        and insulin analogues, such as insulin molecules with minor        differences in the natural amino acid sequence    -   (x) Small molecule mimics of insulin, including, but not limited        to L-783281, TE-17411, and the like,    -   (xi) Na-glucose co-transporter inhibitors, such as T-1095,        T-1095A, phlorizen, and the like,    -   (xii) Amylin agonists which include, but are not limited to        pramlintide, and the like,    -   (xiii) Glucagon antagonists such as AY-279955, and the like.

In addition to antidiabetic agents, bioactive compounds may beanti-obesity agents such as orlistat, a pancreatic lipase inhibitor,which prevents the breakdown and absorption of fat; or sibutramine, anappetite suppressant and inhibitor of the reuptake of serotonin,norepinephrine and dopamine in the brain, growth factors increasing fatmobilization (eg, growth hormone, IGF-1, growth hormone releasingfactor), oxyntomodulin and ghrelin modulators. Other potential bioactiveanti-obesity agents include, but are not limited to,appetite-suppressants acting through adrenergic mechanisms such asbenzphetamine, phenmetrazine, phentermine, diethylpropion, mazindol,sibutramine, phenylpropanolamine or, ephedrine; appetite-suppressantagents acting through serotonergic mechanisms such as quipazine,fluoxetine, sertraline, fenfluramine, or dexfenfluramine;appetite-suppressant agents acting through dopamine mechanisms, eg,apomorphine; appetite-suppressant agents acting through histaminergicmechanisms (eg, histamine mimetics, H3 receptor modulators); enhancersof energy expenditure such as beta-3 adrenergic agonists and stimulatorsof uncoupling protein function; leptin and leptin mimetics (eg,metreleptin); neuropeptide Y antagonists; melanocortin-1, 3 and 4receptor modulators; cholecystokinin agonists; glucagon-like peptide-1(GLP-1) mimetics and analogues (eg, Exendin); androgens (eg,dehydroepiandrosterone and derivatives such as etiocholandione),testosterone, anabolic steroids (eg, oxandrolone), and steroidalhormones; galanin receptor antagonists; cytokine agents such as ciliaryneurotrophic factor; amylase inhibitors; enterostatin agonists/mimetics;orexin/hypocretin antagonists; urocortin antagonists; bombesin agonists;modulators of protein kinase A; corticotropin-releasing factor mimetics;cocaine- and amphetamine-regulated transcript mimetics; calcitonin-generelated peptide mimetics; and fatty acid synthase inhibitors.

In an alternative embodiment, the insulin-hydrogel prodrug compositionaccording to the present invention is combined with a secondbiologically active compound in such way that the insulin-hydrogelprodrug is administered to a patient in need thereof first, followed bythe administration of the second compound. Alternatively, theinsulin-hydrogel composition is administered to a patient in needthereof after another compound has been administered to the samepatient.

Yet another aspect of the present invention is a prodrug of the presentinvention or a pharmaceutical composition of the present invention foruse as a medicament.

Yet another aspect of the present invention is a prodrug of the presentinvention or a pharmaceutical composition of the present invention foruse in a method of treating or preventing diseases or disorders whichcan be treated by insulin.

Such diseases or disorders are e.g. hyperglycemia, pre-diabetes,impaired glucose tolerance, diabetes type I, diabetes type II, syndromeX, obesity, hypertension.

Patients in need of treatment with the long acting insulin compositionsdescribed in the present invention are at high risk of developingcomorbidities. Accordingly, the combination of the long acting insulinof the present with appropriate bioactive compounds may be used, e.g.,for the prevention, delay of progression or treatment of diseases anddisorders selected from the group consisting of hypertension (includingbut not limited to isolated systolic hypertension and familialdyslipidemic hypertension), congestive heart failure, left ventricularhypertrophy, peripheral arterial disease, diabetic retinopathy, maculardegeneration, cataract, diabetic nephropathy, glomerulosclerosis,chronic renal failure, diabetic neuropathy, syndrome X, premenstrualsyndrome, coronary heart disease, angina pectoris, thrombosis,atherosclerosis, myocardial infarction, transient ischemic attacks,stroke, vascular restenosis, hyperglycemia, hyperinsulinemia,hyperlipidemia, hypertriglyceridemia insulin resistance, impairedglucose metabolism, conditions of impaired glucose tolerance, conditionsof impaired fasting plasma glucose, obesity, erectile dysfunction, skinand connective tissue disorders, foot ulcerations and ulcerativecolitis, endothelial dysfunction and impaired vascular compliance.

Prevention, delay of progression or treatment of diseases and disordersselected from the group above can be achieved by combination of the longacting insulin composition of the present invention with at least onebioactive compound selected from the drug classes used for treating saidconditions, including AT₁-receptor antagonists; angiotensin convertingenzyme (ACE) inhibitors; renin inhibitors; beta adrenergic receptorblockers; alpha adrenergic receptor blockers; calcium channel blockers;aldosterone synthase inhibitors; aldosterone receptor antagonists;neutral endopeptidase (NEP) inhibitors; dual angiotensin convertingenzyme/neutral endopetidase (ACE/NEP) inhibitors; an endothelin receptorantagonists; diuretics; statins; nitrates; anti clotting agents;natriuretic peptides; digitalis compounds; PPAR modulators.

In case the biologically active agents; prodrugs, especially hydrogelprodrugs contain one or more acidic or basic groups, the invention alsocomprises their corresponding pharmaceutically or toxicologicallyacceptable salts, in particular their pharmaceutically utilizable salts.Thus, the prodrugs which contain acidic groups can be used according tothe invention, for example, as alkali metal salts, alkaline earth metalsalts or as ammonium salts. More precise examples of such salts includesodium salts, potassium salts, calcium salts, magnesium salts or saltswith ammonia or organic amines such as, for example, ethylamine,ethanolamine, triethanolamine or amino acids. Prodrugs which contain oneor more basic groups, i.e. groups which can be protonated, can bepresent and can be used according to the invention in the form of theiraddition salts with inorganic or organic acids. Examples for suitableacids include hydrogen chloride, hydrogen bromide, phosphoric acid,sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonicacid, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaricacid, lactic acid, salicylic acid, benzoic acid, formic acid, propionicacid, pivalic acid, diethylacetic acid, malonic acid, succinic acid,pimelic acid, fumaric acid, maleic acid, malic acid, sulfaminic acid,phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid,citric acid, adipic acid, and other acids known to the person skilled inthe art. If the prodrugs simultaneously contain acidic and basic groupsin the molecule, the invention also includes, in addition to the saltforms mentioned, inner salts or betaines (zwitterions). The respectivesalts can be obtained by customary methods which are known to the personskilled in the art like, for example by contacting these with an organicor inorganic acid or base in a solvent or dispersant, or by anionexchange or cation exchange with other salts. The present invention alsoincludes all salts of the prodrugs which, owing to low physiologicalcompatibility, are not directly suitable for use in pharmaceuticals butwhich can be used, for example, as intermediates for chemical reactionsor for the preparation of pharmaceutically acceptable salts.

The term “pharmaceutically acceptable” means approved by a regulatoryagency such as the EMEA (Europe) and/or the FDA (US) and/or any othernational regulatory agency for use in animals, preferably in humans.

Yet another aspect of the present invention is a method of treating,controlling, delaying or preventing in a mammalian patient, preferablyin a human, in need of the treatment of one or more conditionscomprising administering to said patient a therapeutically effectiveamount of a prodrug of the present invention or a pharmaceuticalcomposition of the present invention or a pharmaceutically acceptablesalt thereof.

FIG. 1 a: HPLC chromatogram of insulin-linker conjugate 12a

FIG. 1 b: HPLC chromatogram of insulin-linker conjugate 12b

FIG. 2 shows the average plasma insulin concentration of animal 1-10after a single subcutaneous dose of test item 11a containing 6 mginsulin into healthy rats over a 2 week period. (error bars are given as±standard deviation as derived from all 10 animals, t₀ values were taken3 days before dosage.)

FIG. 3: Average plasma insulin concentration of animal 1-8 after asingle subcutaneous dose of test item 11da containing 3 mg insulin intohealthy rats over a period of 13 days. (error bars are given as±standard deviation as derived from all 8 animals, t₀ values were taken1 day before dosage.)

FIG. 4: Plasma insulin concentration (grey squares) and blood glucoselevel (black circles) after a single subcutaneous dose of test item 11dacontaining 6.4 mg insulin into diabetic rats (n=7). (error bars aregiven as ±standard deviation as derived from all 7 animals, t₀ valueswere taken 4 days before dosage.)

FIG. 5: Average plasma insulin level after a single subcutaneous dose of8 mg/kg of test item 11 db into healthy rats during the first 24 hoursafter dosage (burst analysis). 8 rats were divided into 2 groups andblood samples for pharmacokinetics were taken alternating between bothgroups. In neither group was a burst effect perceivable. (Error bars aregiven as ±standard deviation as derived from all animals per group, t₀values were taken 1 day before dosage.)

FIG. 6: Plasma insulin concentration (grey squares) and blood glucoselevel (black circles) during a 4 week period after 3 weekly subcutaneousdoses of 8 mg/kg of test item 11da into diabetic rats (n=8). (error barsare given as ±standard deviation as derived from all 8 animals, t₀values were taken 3 days before dosage.)

FIG. 7: Average plasma insulin concentration of animal 1-8 (animal 1-4and animal 5-8 for 0.3, 1 h, 2, and 4 h value, respectively) after asingle subcutaneous injection of 12 mg/kg insulin formulated in testitem 11dc into healthy rats over a period of 13 days. (error bars aregiven as +/− standard deviation as derived from all 8 animals, t₀ valueswere taken 4 days before dosage)

FIG. 8: Overlay of insulin release and hydrogel degradation ofinsulin-linker-hydrogel 11a. Amount of insulin content ininsulin-linker-hydrogel (triangles) and backbone moieties release(circles) upon incubation of insulin-linker-hydrogel at pH 7.4 and 37°C. is plotted against incubation time.

FIG. 9 shows a graph plotting force versus flow using a 30 G needle.Data points: black squares=ethylene glycol; black triangles=water; blackdots=hydrogel insulin prodrug.

EXAMPLES Materials and Methods

Recombinant human insulin was obtained from Biocon Ltd., Bangalore,India.

Amino 4-arm PEG 5 kDa was obtained from JenKem Technology, Beijing, P.R. China.

N-(3-maleimidopropyl)-21-amino-4,7,10,13,16,19-hexaoxa-heneicosanoicacid NHS ester (Mal-PEG6-NHS) was obtained from Celares GmbH, Berlin,Germany.

2-Chlorotrityl chloride resin, HATU, N-cyclohexyl-carbodiimide-N′-methylpolystyrene, and amino acids were from Merck Biosciences GmbH,Schwalbach/Ts, Germany, if not stated otherwise. Fmoc(NMe)-Asp(OtBu)-OHwas obtained from Bachem AG, Bubendorf, Switzerland.S-Trityl-6-mercaptohexanoic acid was purchased from Polypeptide,Strasbourg, France. Amino acids used were of L configuration if notstated otherwise.

All other chemicals were from Sigma-ALDRICH Chemie GmbH, Taufkirchen,Germany.

Solid phase synthesis was performed on 2-Chlorotrityl chloride (TCP)resin with a loading of 1.3 mmol/g. Syringes equipped with polypropylenefrits were used as reaction vessels.

Loading of the first amino acid to resins was performed according tomanufacturer's instructions.

Fmoc Deprotection:

For Fmoc protecting-group removal, the resin was agitated with 2/2/96(v/v/v) piperidine/DBU/DMF (two times, 10 min each) and washed with DMF(ten times).

Fmoc Deprotection of Fmoc-Aib-Loaded Resins

Fmoc deprotection of immobilized Fmoc-Aib-OH was achieved by stirringthe resin in DMF/piperidine 4/1 (v/v) at 50° C. for 20 min (2 times).

Cleavage Protocol for 2-Chlorotrityl Chloride Resin:

Upon completed synthesis, the resin was washed with DCM, dried in vacuoand treated two times for 30 minutes with 6/4 (v/v) DCM/HFIP. Eluateswere combined, volatiles were removed under a stream of nitrogen and theresulting crude product was purified by RP-HPLC. HPLC fractionscontaining product were combined and lyophilized.

Amine containing products obtained as TFA salts were converted to thecorresponding HCl salts using ion exchange resin (Discovery DSC-SAX,Supelco, USA). This step was performed in case the residual TFA wasexpected to interfere with e.g. a subsequent coupling reaction.

RP-HPLC Purification:

RP-HPLC was done on a 100×20 mm or 100×40 mm C18 ReproSil-Pur 300 ODS-35μ column (Dr. Maisch, Ammerbuch, Germany) connected to a Waters 600HPLC System and Waters 2487 Absorbance detector. Linear gradients ofsolution A (0.1% TFA in H₂O) and solution B (0.1% TFA in acetonitrile)were used. HPLC fractions containing product were lyophilized.

Flash Chromatography

Flash chromatography purifications were performed on an Isolera Onesystem from Biotage AB, Sweden, using Biotage KP-Sil silica cartridgesand n-heptane and ethyl acetate as eluents. Products were detected at254 nm.

For hydrogel beads, syringes equipped with polypropylene frits were usedas reaction vessels or for washing steps.

Analytical Methods

Analytical ultra-performance LC (HPLC) was performed on a Waters Acquitysystem equipped with a Waters BEH300 C18 column (2.1×50 mm, 1.7 μmparticle size) coupled to a LTQ Orbitrap Discovery mass spectrometerfrom Thermo Scientific.

MS of PEG products showed a series of (CH₂CH₂O)_(n) moieties due topolydispersity of PEG staring materials. For easier interpretation onlyone single representative m/z signal is given in the examples. MS ofinsulin conjugates are reported for representative isotopes and refer tothe four-proton adducts [M+4H]⁴⁺.

Size exclusion chromatography (SEC) was performed using an AmershamBioscience AEKTAbasic system equipped with a Superdex200 5/150 GL column(Amersham Bioscience/GE Healthcare) equipped with a 0.45 μm inletfilter, if not stated otherwise. 20 mM sodium phosphate, 140 mM NaCl, pH7.4, was used as mobile phase.

Example 1 Synthesis of Backbone Reagent 1g

Backbone reagent 1g was synthesized from amino 4-arm PEG5000 1aaccording to following scheme:

For synthesis of compound Ib, amino 4-arm PEG5000 1a (MW ca. 5200 g/mol,5.20 g, 1.00 mmol, HCl salt) was dissolved in 20 mL of DMSO (anhydrous).Boc-Lys(Boc)-OH (2.17 g, 6.25 mmol) in 5 mL of DMSO (anhydrous), EDC HCl(1.15 g, 6.00 mmol), HOBt.H₂O (0.96 g, 6.25 mmol), and collidine (5.20mL, 40 mmol) were added. The reaction mixture was stirred for 30 min atRT.

The reaction mixture was diluted with 1200 mL of dichloromethane andwashed with 600 mL of 0.1 N H₂SO₄ (2×), brine (1×), 0.1 M NaOH (2×), and1/1 (v/v) brine/water (4×). Aqueous layers were reextracted with 500 mLof DCM. Organic phases were dried over Na₂SO₄, filtered and evaporatedto give 6.3 g of crude product 1b as colorless oil. Compound 1b waspurified by RP-HPLC.

Yield 3.85 g (59%) colorless glassy product 1b.

MS: m/z 1294.4=[M+5H]⁵⁺ (calculated=1294.6).

Compound 1c was obtained by stirring of 3.40 g of compound 1b (0.521mmol) in 5 mL of methanol and 9 mL of 4 N HCl in dioxane at RT for 15min. Volatiles were removed in vacuo. The product was used in the nextstep without further purification.

MS: m/z 1151.9=[M+5H]⁵⁺ (calculated=1152.0).

For synthesis of compound 1d, 3.26 g of compound 1c (0.54 mmol) weredissolved in 15 mL of DMSO (anhydrous). 2.99 g Boc-Lys(Boc)-OH (8.64mmol) in 15 mL DMSO (anhydrous), 1.55 g EDC HCl (8.1 mmol), 1.24 gHOBt.H₂O (8.1 mmol), and 5.62 mL of collidine (43 mmol) were added. Thereaction mixture was stirred for 30 min at RT.

Reaction mixture was diluted with 800 mL DCM and washed with 400 mL of0.1 N H₂SO₄ (2×), brine (1×), 0.1 M NaOH (2×), and 1/1 (v/v) brine/water(4×). Aqueous layers were reextracted with 800 mL of DCM. Organic phaseswere dried with Na₂SO₄, filtered and evaporated to give a glassy crudeproduct.

Product was dissolved in DCM and precipitated with cooled (−18° C.)diethylether. This procedure was repeated twice and the precipitate wasdried in vacuo.

Yield: 4.01 g (89%) colorless glassy product 1d, which was used in thenext step without further purification.

MS: m/z 1405.4=[M+6H]⁶⁺ (calculated=1405.4).

Compound 1e was obtained by stirring a solution of compound 1d (3.96 g,0.47 mmol) in 7 mL of methanol and 20 mL of 4 N HCl in dioxane at RT for15 min. Volatiles were removed in vacuo. The product was used in thenext step without further purification.

MS: m/z 969.6=[M+7H]⁷⁺ (calculated=969.7).

For the synthesis of compound 1f, compound 1e (3.55 g, 0.48 mmol) wasdissolved in 20 mL of DMSO (anhydrous). Boc-Lys(Boc)-OH (5.32 g, 15.4mmol) in 18.8 mL of DMSO (anhydrous), EDC HCl (2.76 g, 14.4 mmol),HOBt.H₂O (2.20 g, 14.4 mmol), and 10.0 mL of collidine (76.8 mmol) wereadded. The reaction mixture was stirred for 60 min at RT.

The reaction mixture was diluted with 800 mL of DCM and washed with 400mL of 0.1 N H₂SO₄ (2×), brine (1×), 0.1 M NaOH (2×), and 1/1 (v/v)brine/water (4×). Aqueous layers were reextracted with 800 mL of DCM.Organic phases were dried over Na₂SO₄, filtered and evaporated to givecrude product 1f as colorless oil.

Product was dissolved in DCM and precipitated with cooled (−18° C.)diethylther. This step was repeated twice and the precipitate was driedin vacuo.

Yield 4.72 g (82%) colourless glassy product 1f which was used in thenext step without further purification.

MS: m/z 1505.3=[M+8H]⁸⁺ (calculated=1505.4).

Backbone reagent 1g was obtained by stirring a solution of compound 1f(MW ca 12035 g/mol, 4.72 g, 0.39 mmol) in 20 mL of methanol and 40 mL of4 N HCl in dioxane at RT for 30 min. Volatiles were removed in vacuo.

Yield 3.91 g (100%), glassy product backbone reagent 1g.

MS: m/z 977.2=[M+9H]⁹⁺ (calculated=977.4).

Alternative Synthetic Route for 1g

For synthesis of compound 1b, to a suspension of 4-Arm-PEG5000tetraamine (1a) (50.0 g, 10.0 mmol) in 250 mL of iPrOH (anhydrous),boc-Lys(boc)-OSu (26.6 g, 60.0 mmol) and DIEA (20.9 mL, 120 mmol) wereadded at 45° C. and the mixture was stirred for 30 min.

Subsequently, n-propylamine (2.48 mL, 30.0 mmol) was added. After 5 minthe solution was diluted with 1000 mL of MTBE and stored overnight at−20° C. without stirring. Approximately 500 mL of the supernatant weredecanted off and discarded. 300 mL of cold MTBE were added and after 1min shaking the product was collected by filtration through a glassfilter and washed with 500 mL of cold MTBE. The product was dried invacuo for 16 h.

Yield: 65.6 g (74%) 1b as a white lumpy solid

MS: m/z 937.4=[M+7H]⁷⁺ (calculated=937.6).

Compound 1c was obtained by stirring of compound 1b from the previousstep (48.8 g, 7.44 mmol) in 156 mL of 2-propanol at 40° C. A mixture of196 mL of 2-propanol and 78.3 mL of acetylchloride was added understirring within 1-2 min. The solution was stirred at 40° C. for 30 minand cooled to −30° C. overnight without stirring. 100 mL of cold MTBEwere added, the suspension was shaken for 1 min and cooled for 1 h at−30° C. The product was collected by filtration through a glass filterand washed with 200 mL of cold MTBE. The product was dried in vacuo for16 h.

Yield: 38.9 g (86%) 1c as a white powder

MS: m/z 960.1=[M+6H]⁶⁺ (calculated=960.2).

For synthesis of compound 1d, to a suspension of 1c from the previousstep (19.0 g, 3.14 mmol) in 80 ml 2-propanol boc-Lys(boc)-OSu (16.7 g,37.7 mmol) and DIEA (13.1 mL, 75.4 mmol) were added at 45° C. and themixture was stirred for 30 min at 45° C. Subsequently, n-propylamine(1.56 mL, 18.9 mmol) was added. After 5 min the solution wasprecipitated with 600 mL of cold MTBE and centrifuged (3000 min⁻¹, 1min) The precipitate was dried in vacuo for 1 h and dissolved in 400 mLTHF. 200 mL of diethyl ether were added and the product was cooled to−30° C. for 16 h without stirring. The suspension was filtered through aglass filter and washed with 300 mL cold MTBE. The product was dried invacuo for 16 h.

Yield: 21.0 g (80%) 1d as a white solid

MS: m/z 1405.4=[M+6H]⁶⁺ (calculated=1405.4).

Compound 1e was obtained by dissolving compound 1d from the previousstep (15.6 g, 1.86 mmol) in 3 N HCl in methanol (81 mL, 243 mmol) andstirring for 90 min at 40° C. 200 mL of MeOH and 700 mL of iPrOH wereadded and the mixture was stored for 2 h at −30° C. For completeness ofcrystallization, 100 mL of MTBE were added and the suspension was storedat −30° C. overnight. 250 mL of cold MTBE were added, the suspension wasshaken for 1 min and filtered through a glass filter and washed with 100mL of cold MTBE. The product was dried in vacuo.

Yield: 13.2 g (96%) 1e as a white powder

MS: m/z 679.1=[M+10H]¹⁰⁺ (calculated=679.1).

For the synthesis of compound 1f, to a suspension of 1e from theprevious step, (8.22 g, 1.12 mmol) in 165 ml 2-propanol boc-Lys(boc)-OSu(11.9 g, 26.8 mmol) and DIEA (9.34 mL, 53.6 mmol) were added at 45° C.and the mixture was stirred for 30 min. Subsequently, n-propylamine(1.47 mL, 17.9 mmol) was added. After 5 min the solution was cooled to−18° C. for 2 h, then 165 mL of cold MTBE were added, the suspension wasshaken for 1 min and filtered through a glass filter. Subsequently, thefilter cake was washed with 4×200 mL of cold MTBE/iPrOH 4:1 and 1×200 mLof cold MTBE. The product was dried in vacuo for 16 h.

Yield: 12.8 g, MW (90%) 1f as a pale yellow lumpy solid

MS: m/z 1505.3=[M+8H]⁸⁺ (calculated=1505.4).

Backbone reagent 1g was obtained by dissolving 4ArmPEG5kDa(-LysLys₂Lys₄(boc)₈)₄ (1f) (15.5 g, 1.29 mmol) in 30 mL of MeOH andcooling to 0° C. 4 N HCl in dioxane (120 mL, 480 mmol, cooled to 0° C.)was added within 3 min and the ice bath was removed. After 20 min, 3 NHCl in methanol (200 mL, 600 mmol, cooled to 0° C.) was added within 15min and the solution was stirred for 10 min at room temperature. Theproduct solution was precipitated with 480 mL of cold MTBE andcentrifuged at 3000 rpm for 1 min. The precipitate was dried in vacuofor 1 h and redissolved in 90 mL of MeOH, precipitated with 240 mL ofcold MTBE and the suspension was centrifuged at 3000 rpm for 1 min. Theproduct 1g was dried in vacuo

Yield: 11.5 g (89%) as pale yellow flakes.

MS: m/z 1104.9=[M+8H]⁸⁺ (calculated=1104.9).

Example 2 Synthesis of Crosslinker Reagent 2d

Crosslinker reagent 2d was prepared from adipic acid mono benzyl ester(English, Arthur R. et al., Journal of Medicinal Chemistry, 1990, 33(1),344-347) and PEG2000 according to the following scheme:

A solution of PEG 2000 (2a) (11.0 g, 5.5 mmol) and benzyl adipatehalf-ester (4.8 g, 20.6 mmol) in dichloromethane (90.0 mL) was cooled to0° C. Dicyclohexylcarbodiimide (4.47 g, 21.7 mmol) was added followed bya catalytic amount of DMAP (5 mg) and the solution was stirred andallowed to reach room temperature overnight (12 h). The flask was storedat +4° C. for 5 h. The solid was filtered and the solvent completelyremoved by destillation in vacuo. The residue was dissolved in 1000 mL1/1(v/v) diethyl ether/ethyl acetate and stored at RT for 2 hours whilea small amount of a flaky solid was formed. The solid was removed byfiltration through a pad of Celite®. The solution was stored in atightly closed flask at −30° C. in the freezer for 12 h untilcrystallisation was complete. The crystalline product was filteredthrough a glass frit and washed with cooled diethyl ether (−30° C.). Thefilter cake was dried in vacuo. Yield: 11.6 g (86%) 2b as a colorlesssolid. The product was used without further purification in the nextstep.

MS: m/z 813.1=[M+3H]³⁺ (calculated=813.3)

In a 500 mL glass autoclave PEG2000-bis-adipic acid-bis-benzyl ester 2b(13.3 g, 5.5 mmol) was dissolved in ethyl acetate (180 mL) and 10%Palladium on charcoal (0.4 g) was added. The solution was hydrogenatedat 6 bar, 40° C. until consumption of hydrogen had ceased (5-12 h).Catalyst was removed by filtration through a pad of Celite® and thesolvent was evaporated in vacuo. Yield: 12.3 g (quantitative) 2c asyellowish oil. The product was used without further purification in thenext step.

MS: m/z 753.1=[M+3H]³⁺ (calculated=753.2)

A solution of PEG2000-bis-adipic acid half ester 2c (9.43 g, 4.18 mmol),N-hydroxysuccinimide (1.92 g, 16.7 mmol) and dicyclohexylcarbodiimide(3.44 g, 16.7 mmol) in 75 mL of DCM (anhydrous) was stirred over nightat room temperature. The reaction mixture was cooled to 0° C. andprecipitate was filtered off. DCM was evaporated and the residue wasrecystallized from THF.

Yield: 8.73 g (85%) crosslinker reagent 2d as colorless solid.

MS: m/z 817.8=[M+3H]³⁺ (calculated=817.9 g/mol).

Example 3 Preparation of Hydrogel Beads (3) and (3a) Containing FreeAmino Groups

A solution of 275 mg 1g and 866 mg 2d in 14 mL DMSO was added to asolution of 100 mg Arlacel P135 (Croda International Plc) in 60 mLheptane. The mixture was stirred at 700 rpm with a custom metal stirrerfor 10 min at 25° C. to form a suspension. 1.0 mLN,N,N′,N′-tetramethyl-ethylene-diamine was added to effectpolymerization. After 2 h, the stirrer speed was reduced to 400 rpm andthe mixture was stirred for additional 16 h. 1.5 mL of acetic acid wereadded and then after 10 min 50 mL of water were added. After 5 min, thestirrer was stopped and the aqueous phase was drained.

For bead size fractionation, the water-hydrogel suspension waswet-sieved on 75, 50, 40, 32 and 20 μm mesh steel sieves. Bead fractionsthat were retained on the 32, 40, and 50 μm sieves were pooled andwashed 3 times with water, 10 times with ethanol and dried for 16 h at0.1 mbar to give 3 as a white powder.

3a was prepared as described for 3 except for the use of 1200 mg 1g,3840 mg 2d, 28.6 ml DMSO, 425 mg Arlacel P135, 100 mL heptane and 4.3 mlTMEDA. For workup, 6.6 ml acetic acid were added and then after 10 min50 mL of water and 50 mL of saturated aqueous sodium chloride solutionwere added.

Amino group content of hydrogel was determined by conjugation of afmoc-amino acid to the free amino groups on the hydrogel and subsequentfmoc-determination as described by Gude, M., J. Ryf, et al. (2002)Letters in Peptide Science 9(4): 203-206.

The amino group content of 3 and 3a was determined to be between 0.11and 0.16 mmol/g.

Example 4 Preparation of Maleimide Functionalized Hydrogel Beads (4) and(4a) and (4aa) and Determination of Maleimide Substitution

A solution of 600 mg Mal-PEG6-NHS (1.0 mmol) in 4.5 mL 2/1 (v/v)acetonitrile/water was added to 200 mg dry hydrogel beads 3. 500 μLsodium phosphate buffer (pH 7.4, 0.5 M) was added and the suspension wasagitated for 30 min at room temperature. Beads 4 were washed five timeseach with 2/1 (v/v) acetonitrile/water, methanol and 1/1/0.001 (v/v/v/)acetonitrile/water/TFA. 4a was synthesized as described above except forthe use of 3a instead of 3.

Alternatively, hydrogel beads 3a were pre-washed with 99/1 (v/v)DMSO/DIEA, washed with DMSO and incubated for 45 min with a solution ofMal-PEG6-NHS (2.0 eq relative to theoretical amount of amino groups onhydrogel) in DMSO. Beads 4aa were washed two times with DMSO and threetimes with pH 3.0 succinate (20 mM, 1 mM EDTA, 0.01% Tween-20). Thesample was incubated in pH 6.0 sodium phosphate (50 mM, 50 mMethanolamine, 0.01% Tween-20) for 1 h at RT and washed five times withpH 3.0 sodium succinate (20 mM, 1 mM EDTA, 0.01% Tween-20).

For determination of maleimide content, an aliquot of hydrogel beads 4,4a, or 4aa, respectively, was lyophilized and weighed out. Anotheraliquot of hydrogel beads 4, 4a or 4aa, respectively, was reacted withexcess mercaptoethanol (in 50 mM sodium phosphate buffer, 30 min at RT),and mercaptoethanol consumption was detected by Ellman test (Ellman, G.L. et al., Biochem. Pharmacol., 1961, 7, 88-95). Maleimide content wasdetermined to be between 0.11 and 0.13 mmol/g dry hydrogel.

Example 5 Synthesis of Linker Reagent 5d

Linker reagent 5d was synthesized according to the following scheme:

Synthesis of Linker Reagent Intermediate 5a:

4-Methoxytrityl chloride (3 g, 9.71 mmol) was dissolved in DCM (20 mL)and added dropwise to a solution of ethylenediamine (6.5 mL, 97.1 mmol)in DCM (20 mL). After two hours the solution was poured into diethylether (300 mL) and washed three times with 30/1 (v/v) brine/0.1 M NaOHsolution (50 ml each) and once with brine (50 mL). The organic phase wasdried over Na₂SO₄ and volatiles were removed under reduced pressure toobtain the Mmt-protected intermediate (3.18 g, 9.56 mmol).

The Mmt-protected intermediate (3.18 g, 9.56 mmol) was dissolved inanhydrous DCM (30 mL). 6-(Tritylmercapto)-hexanoic acid (4.48 g, 11.47mmol), PyBOP (5.67 g, 11.47 mmol) and DIEA (5.0 mL, 28.68 mmol) wereadded and the mixture was agitated for 30 min at RT. The solution wasdiluted with diethyl ether (250 mL) and washed three times with 30/1(v/v) brine/0.1 M NaOH solution (50 mL each) and once with brine (50mL). The organic phase was dried over Na₂SO₄ and volatiles were removedunder reduced pressure. 5a was purified by flash chromatography.

Yield: 5.69 g (8.09 mmol).

MS: m/z 705.4=[M+H]⁺ (calculated=705.0).

Synthesis of Linker Reagent Intermediate 5b:

To a solution of 5a (3.19 g, 4.53 mmol) in anhydrous THF (50 mL) wasadded BH₃.THF (1 M solution, 8.5 mL, 8.5 mmol) and the solution wasstirred for 16 hours at RT. Further BH₃.THF (1 M solution, 14 mL, 14mmol) was added and stirred for 16 hours at RT. The reaction wasquenched by addition of methanol (8.5 mL), N,N-dimethyl-ethylenediamine(3 mL, 27.2 mmol) was added and the solution was heated to reflux andstirred for three hours. The mixture was diluted with ethyl acetate (300mL) at RT, washed with saturated, aqueous Na₂CO₃ solution (2×100 mL) andsaturated, aqueous NaHCO₃ solution (2×100 mL). The organic phase wasdried over Na₂SO₄ and volatiles were evaporated at reduced pressure toobtain the crude amine intermediate (3.22 g).

The amine intermediate was dissolved in DCM (5 mL), Boc₂O (2.97 g, 13.69mmol) dissolved in DCM (5 mL) and DIEA (3.95 mL, 22.65 mmol) were addedand the mixture was agitated at RT for 30 min. The mixture was purifiedby flash chromatography to obtain the crude Boc- and Mmt-protectedintermediate (3 g).

MS: m/z 791.4=[M+H]⁺, 519.3=[M-Mmt+H]⁺ (calculated=791.1).

0.4 M aqueous HCl (48 mL) was added to a solution of the Boc- andMmt-protected intermediate in acetonitrile (45 mL). The mixture wasdiluted with acetonitrile (10 mL) and stirred for one hour at RT.Subsequently, the pH value of the reaction mixture was adjusted to 5.5by addition of 5 M NaOH solution, acetonitrile was removed under reducedpressure and the aqueous solution was extracted with DCM (4×100 mL). Thecombined organic phases were dried over Na₂SO₄ and volatiles wereremoved under reduced pressure. Crude 5b was used without furtherpurification.

Yield: 2.52 g (3.19 mmol).

MS: m/z 519.3=[M+H]⁺ (MW calculated=518.8 g/mol).

Synthesis of Linker Reagent Intermediate 5c:

5b (780 mg, 0.98 mmol, ˜65% purity) and NaCNBH₃ (128 mg, 1.97 mmol) weredissolved in anhydrous methanol (13 mL). A solution of2,4-dimethoxybenzaldehyde (195 mg, 1.17 mmol) in DCM (2 mL) was added,and the mixture was stirred for 2 h at RT. The solvents were evaporatedunder reduced pressure, and the crude product was dissolved in DCM andwashed with saturated NaCO₃ solution. The aqueous phase was extractedthree times with DCM, and the combined organic phases were washed withbrine, dried over MgSO₄ and concentrated under reduced pressure. 5c waspurified by flash chromatography using DCM and MeOH as eluents.

Yield: 343 mg (0.512 mmol).

MS: m/z 669.37=[M+H]⁺, (calculated=669.95).

Synthesis of Linker Reagent 5d:

Fmoc-Aib-loaded TCP resin (980 mg, ˜0.9 mmol) was deprotected withDMF/piperidine, washed with DMF (5 times) and DCM (6 times) and dried invacuo. The resin was treated with a solution of p-nitrophenylchloroformate (364 mg, 1.81 mmol) and collidine (3984, 3.0 mmol) inanhydrous THF (6 mL) and shaken for 30 min. The reagent solution wasremoved by filtration and the resin was washed with THF (5 times) beforea solution of amine 5c (490 mg, 0.7 mmol) and DIEA (1.23 mL, 7.1 mmol)in anhydrous THF (6 mL) was added. After shaking for 18 h at RT, thereagent solution was removed by filtration and the resin was washed withDCM (5 times). The linker reagent was cleaved from the resin andpurified by RP-HPLC. Product fractions were brought to pH 6 by additionof sat. aq. NaHCO₃ and concentrated under reduced pressure. Theresulting slurry was partitioned between saturated aqueous NaCl and DCM,and the aqueous layer was extracted with DCM. The combined organicfractions were concentrated to dryness to afford linker reagent 5d.

Yield: 230 mg, (0.29 mmol).

MS m/z 798.41=[M+H]⁺, (calculated=798.1).

Example 6 Synthesis of Linker Reagent 6c

Linker reagent 6c was synthesized according to the following scheme:

Synthesis of Amine 6a:

Triphenylmethanethiol (11.90 g, 43.08 mmol) was suspended in DMSO (40mL). DBU (7.41 mL, 49.55 mmol) and 6-bromohexylphthalimide (13.32 g,42.94 mmol) were added, and the mixture was allowed to react forapproximately 15 min. The reaction mixture was partitioned between ethylacetate (700 mL) and 0.1 M HCl (200 mL). The aqueous phase was extractedwith ethyl acetate (3×50 mL), and the combined organic fractions werewashed with NaHCO₃ sat. (80 mL) and brine (80 mL), dried over MgSO₄,filtered and concentrated. The crude yellow oil was recrystallized fromn-heptane/ethyl acetate. The intermediate6-(S-Trityl-)mercaptohexylphthalimide was obtained as a white solid(13.3 g, 26.4 mmol, 62%).

6-(S-Trityl-)mercaptohexylphthalimide (14.27 g, 28.2 mmol) was suspendedin ethanol (250 mL). Hydrazine hydrate (3.45 mL, 70.5 mmol) was added,and the mixture was heated to reflux for 2 h. The mixture was filteredand the filtrate was concentrated in vacuo. Chloroform (180 mL) wasadded to the residual oil and the resulting suspension was stirred atroom temperature for 1.5 h. The mixture was filtered, and the filtratewas extracted with water (60 mL) and brine (60 mL), dried over MgSO₄ andconcentrated to yield crude 6-(tritylmercapto)-hexylamine (10.10 g,26.87 mmol, 95%).

MS: m/z 376.22=[M+H]⁺, (calculated=376.20).

DIEA (1.41 mL, 8.11 mmol) and n-butyl chloroformate (908 μL, 7.14 mmol,in 1 mL THF) were added to a cooled (0° C.) solution of6-(tritylmercapto)-hexylamine (2.44 g, 6.49 mmol) in THF (50 mL). LiAlH₄(1 M in THF, 9.74 mL, 9.47 mmol) was added after 30 min, and the mixturewas heated to reflux for 90 min. Addition of water, 3.75 M aq. NaOH andwater led to the formation of a precipitate which was removed from themixture by filtration. The filtrate was concentrated in vacuo to obtain6a.

Yield: 2.41 g (6.20 mmol).

MS: m/z 390.22=[M+H]⁺, (calculated=390.22).

Synthesis of Linker Reagent Intermediate 6b:

To a solution of 6a (2.1 g, 5.31 mmol) was added 2-bromoethylphthalimide(1.96 g, 7.7 mmol) and K₂CO₃ (1.09 g, 7.9 mmol) and the mixture washeated to reflux for 6 h. After filtration and concentration, the crudemixture was partitioned between ethyl acetate and saturated aqueousNaHCO₃. The crude intermediate(2-(N-methyl-N-(6-tritylmercaptohexyl-)amino-)ethyl)phthalimide waspurified by flash chromatography.

Yield: 1.23 g (2.18 mmol).

MS: m/z: 563.27=[M+H]⁺, (calculated=563.27).

To a solution of(2-(N-methyl-N-(6-tritylmercaptohexyl-)amino-)ethyl)phthalimide (672 mg,1.19 mmol) in ethanol (12 mL) was added hydrazine monohydrate (208 μL,4.17 mmol), and the mixture was heated to reflux for 1 h. The reactionmixture was filtered, concentrated andN-(2-aminoethyl-)-N-methyl-N-(6-tritylmercaptohexyHannine purified byRP-HPLC.

Yield: 624 mg (0.944 mmol).

MS: m/z 433.27=[M+H]⁺, (calculated=433.26).

To a solution ofN-(2-aminoethyl-)-N-methyl-N-(6-tritylmercaptohexyl-)amine (151 mg,0.229 mmol) and NaCNBH₃ (30 mg, 0.463 mmol) in anhydrous MeOH (6 mL) wasadded a solution of 2,4-dimethoxybenzaldehyde in anhydrous CH₂Cl₂ (0.6μL). After stirring for 1 h at RT, the reaction mixture wasconcentrated, redissolved in 2 mL water/acetonitrile 1/9 (v/v) and 6bpurified by RP-HPLC.

Yield: 177 mg (0.219 mmol).

MS: m/z 583.33=[M+H]⁺, (calculated=583.33).

Synthesis of Linker Reagent 6c

Linker reagent 6c was prepared from Fmoc-Aib-loaded resin (704 mg, ˜0.6mmol) as described for 5d, except for the use of amine 6b (as TFA salt,430 mg, 0.53 mmol) instead of 5c.

Yield: 285 mg, (0.330 mmol).

MS: m/z 712.37=[M+H]⁺, (calculated=712.37).

Example 7 Synthesis of Linker Reagent 7f

Linker reagent 7f was synthesized according to the following scheme:

To a cooled (0° C.) solution of N-Methyl-N-boc-ethylendiamine (0.5 mL,2.79 mmol) and NaCNBH₃ (140 mg, 2.23 mmol) in MeOH (10 mL) and aceticacid (0.5 mL) was added a solution of 2,4,6-trimethoxybenzaldehyde(0.547 mg, 2.79 mmol) in EtOH (10 mL). The mixture was stirred at RT for2 h, acidified with 2 M HCl (1 mL) and neutralized with saturatedaqueous Na₂CO₃ (50 mL). Evaporation of all volatiles, DCM extraction ofthe resulting aqueous slurry and concentration of the organic fractionsyielded N-Methyl-N-boc-N′-tmob-ethylendiamine (7a) as a crude oil whichwas purified by RP-HPLC.

Yield: 593 mg (1.52 mmol)

MS: m/z 377.35=[M+Na]⁺, (calculated=377.14).

N-Fmoc-N-Me-Asp(OtBu)-OH (225 mg, 0.529 mmol) was dissolved in DMF (3mL) and 7a (300 mg, 0.847 mmol), HATU (201 mg, 0.529 mmol), andcollidine (0.48 mL, 3.70 mmol) were added. The mixture was stirred at RTfor 2 h to yield 7b. For fmoc deprotection, piperidine (0.22 mL, 2.16mmol) was added and stirring was continued for 1 h. Acetic acid (1 mL)was added, and 7c was purified by RP-HLPC.

Yield: 285 mg (0.436 mmol as TFA salt)

MS: m/z 562.54=[M+Na]⁺, (calculated=562.67).

6-Tritylmercaptohexanoic acid (0.847 g, 2.17 mmol) was dissolved inanhydrous DMF (7 mL). HATU (0.825 g, 2.17 mmol), and collidine (0.8 mL,6.1 mmol) and 7c (0.78 g, 1.44 mmol) were added. The reaction mixturewas stirred for 60 min at RT, acidified with AcOH (1 mL) and purified byRP-HPLC. Product fractions were neutralized with saturated aqueousNaHCO₃ and concentrated. The remaining aqueous phase was extracted withDCM and 7d was isolated upon evaporation of the solvent.

Yield: 1.4 g (94%)

MS: m/z 934.7=[M+Na]⁺, (calculated=934.5).

To a solution of 7d (1.40 mg, 1.53 mmol) in MeOH (12 mL) and H₂O (2 mL)was added LiOH (250 mg, 10.4 mmol) and the reaction mixture was stirredfor 14 h at 70° C. The mixture was acidified with AcOH (0.8 mL) and 7ewas purified by RP-HPLC. Product fractions were neutralized withsaturated aqueous NaHCO₃ and concentrated. The aqueous phase wasextracted with DCM and 7e was isolated upon evaporation of the solvent.

Yield: 780 mg (60%)

MS: m/z 878.8=[M+Na]⁺, (calculated=878.40).

To a solution of 7e (170 mg, 0.198 mmol) in anhydrous DCM (4 mL) wereadded DCC (123 mg, 0.59 mmol) and N-hydroxy-succinimide (114 mg, 0.99mmol), and the reaction mixture was stirred at RT for 1 h. The mixturewas filtered, and the filtrate was acidified with 0.5 mL AcOH and 7fpurified by RP-HPLC. Product fractions were neutralized with saturatedaqueous NaHCO₃ and concentrated. The remaining aqueous phase wasextracted with DCM and 7f was isolated upon evaporation of the solvent.

Yield: 154 mg (0.161 mmol)

MS: m/z 953.4=[M+H]⁺, (calculated=953.43).

Alternatively, linker reagent 7f was synthesized according to thefollowing procedure:

Alternative reaction scheme:

To a solution of N-Methyl-N-boc-ethylenediamine (2 g, 11.48 mmol) andNaCNBH₃ (819 mg, 12.63 mmol) in MeOH (20 mL) was added2,4,6-trimethoxybenzaldehyde (2.08 mg, 10.61 mmol) portion wise. Themixture was stirred at RT for 90 min, acidified with 3 M HCl (4 mL) andstirred further 15 min. The reaction mixture was added to saturatedNaHCO₃ solution (200 mL) and extracted 5× with CH₂Cl₂. The combinedorganic phases were dried over Na₂SO₄ and the solvents were evaporatedin vacuo. The resulting N-Methyl-N-boc-N′-tmob-ethylenediamine (7a) wascompletely dried in high vacuum and used in the next reaction stepwithout further purification.

Yield: 3.76 g (11.48 mmol, 89% purity, 7a: double Tmob protectedproduct=8:1)

MS: m/z 355.22=[M+H]⁺, (calculated=354.21).

To a solution of 7a (2 g, 5.65 mmol) in CH₂Cl₂ (24 ml) COMU (4.84 g,11.3 mmol), N-Fmoc-N-Me-Asp(OBn)-OH (2.08 g, 4.52 mmol) and collidine(2.65 mL, 20.34 mmol) were added. The reaction mixture was stirred for 3h at RT, diluted with CH₂Cl₂ (250 mL) and washed 3× with 0.1 M H₂SO₄(100 ml) and 3× with brine (100 ml). The aqueous phases were reextracted with CH₂Cl₂ (100 ml). The combined organic phases were driedover Na₂SO₄, filtrated and the residue concentrated to a volume of 24mL. 7g was purified using flash chromatography.

Yield: 5.31 g (148%, 6.66 mmol)

MS: m/z 796.38=[M+H]⁺, (calculated=795.37).

To a solution of 7g [5.31 g, max. 4.51 mmol ref. toN-Fmoc-N-Me-Asp(OBn)-OH] in THF (60 mL) DBU (1.8 mL, 3% v/v) was added.The solution was stirred for 12 min at RT, diluted with CH₂Cl₂ (400 ml)and washed 3× with 0.1 M H₂SO₄ (150 ml) and 3× with brine (150 ml). Theaqueous phases were re extracted with CH₂Cl₂ (100 ml). The combinedorganic phases were dried over Na₂SO₄ and filtrated. 7h was isolatedupon evaporation of the solvent and used in the next reaction withoutfurther purification.

MS: m/z 574.31=[M+H]⁺, (calculated=573.30).

7h (5.31 g, 4.51 mmol, crude) was dissolved in acetonitrile (26 mL) andCOMU (3.87 g, 9.04 mmol), 6-Tritylmercaptohexanoic acid (2.12 g, 5.42mmol) and collidine (2.35 mL, 18.08 mmol) were added. The reactionmixture was stirred for 4 h at RT, diluted with CH₂Cl₂ (400 ml) andwashed 3× with 0.1 M H₂SO₄ (100 ml) and 3× with brine (100 ml). Theaqueous phases were re extracted with CH₂Cl₂ (100 ml). The combinedorganic phases were dried over Na₂SO₄, filtrated and 7i was isolatedupon evaporation of the solvent. Product 7i was purified using flashchromatography.

Yield: 2.63 g (62%, 94% purity)

MS: m/z 856.41=[M+H]⁺, (calculated=855.41).

To a solution of 7i (2.63 g, 2.78 mmol) in i-PrOH (33 mL) and H₂O (11mL) was added LiOH (267 mg, 11.12 mmol) and the reaction mixture wasstirred for 70 min at RT. The mixture was diluted with CH₂Cl₂ (200 ml)and washed 3× with 0.1 M H₂SO₄ (50 ml) and 3× with brine (50 ml). Theaqueous phases were re-extracted with CH₂Cl₂ (100 ml). The combinedorganic phases were dried over Na₂SO₄, filtrated and 7e was isolatedupon evaporation of the solvent. 7j was purified using flashchromatography.

Yield: 2.1 g (88%)

MS: m/z 878.4=[M+Na]⁺, (calculated=878.40).

To a solution of 7e (170 mg, 0.198 mmol) in anhydrous DCM (4 mL) wereadded DCC (123 mg, 0.59 mmol), and a catalytic amount of DMAP. After 5min N-hydroxy-succinimide (114 mg, 0.99 mmol) was added and the reactionmixture was stirred at RT for 1 h. The reaction mixture was filtered,the solvent was removed in vacuo and the residue was taken up in 90%acetonitrile plus 0.1% TFA (3.4 ml). The crude mixture was purified byRP-HPLC. Product fractions were neutralized with 0.5 M pH 7.4 phosphatebuffer and concentrated. The remaining aqueous phase was extracted withDCM and 7f was isolated upon evaporation of the solvent.

Yield: 154 mg (81%)

MS: m/z 953.4=[M+H]⁺, (calculated=953.43).

Example 8 Synthesis of N^(αA1)-Insulin-Linker Conjugates 8b and 8c

Synthesis of Protected Insulin Linker Conjugate 8a

Linker reagent 5d was dissolved in DCM (20 mg/mL) and activated withN-cyclohexyl-carbodiimide-N′-methyl polystyrene-resin (1.9 mmol/g, 10eq.) for 1 h. The solution of the activated linker reagent was added toa solution of insulin (1.2 eq.) and DIEA (3.5 eq.) in DMSO (100 mginsulin/mL), and the mixture was shaken at RT for 45 min. The solutionwas acidified with acetic acid, the DCM was evaporated under reducedpressure, and N^(αA1)-conjugated protected insulin-linker conjugate 8awas purified by RP-HPLC.

Lyophilized 8a was treated with a mixture of 90/10/2/2 (v/v/v/v)HFIP/TFA/water/triethylsilane (2 mL/100 mg of 8a) for 45 min at RT. Thereaction mixture was diluted with water, and all volatiles were removedunder a stream of nitrogen. N^(αA1)-conjugated insulin-linker conjugate8b was purified by RP-HPLC.

8b:

Yield: 139 mg (0.023 mmol) from 62 mg (0.078 mmol) linker 5d

MS: m/z 1524.45=[M+4H]⁴⁺ (calculated=1524.75).

N^(αA1)-conjugated insulin-linker conjugate 8c was synthesized asdescribed for 8b except for the use of 6c (72 mg, 0.101 mmol) instead of5d.

8c:

Yield: 237 mg (0.039 mmol)

MS: m/z 1528.23=[M+4H]⁺ (calculated=1528.28).

Example 9 Synthesis of N^(αB1)-Insulin-Linker Conjugate 9

Double-protected N^(α)-boc-Gly^(A1)-N^(ε)-boc-Lys^(B29)-insulin wasprepared as described previously (J. Markussen, J. Halstrøm, F. C.Wiberg, L. Schäffer, J. Biol. Chem. 1991, 266, 18814-18818). Linkerreagent 5d (0.04 mmol) was dissolved in DCM (0.5 mL) and activated withN-cyclohexyl-carbodiimide-N′-methyl polystyrene resin (0.205 mmol) at RTfor 2 h. The resulting solution of the activated linker reagent wasadded to a solution of bis-boc-protected insulin (24 mg, 0.004 mmol) andDIEA (5 μL, 0.0229 mmol) and shaken at RT for 1 h. The reaction mixturewas acidified with 100 μL of acetic acid and protected insulin-linkerconjugate was purified by RP-HPLC.

Yield: 5 mg (0.00075 mmol).

MS: m/z 1660.27=[M+4H]⁺ (calculated=1660.43).

Lyophilized protected insulin-linker conjugate was treated with 1 mL90/10/2/2 (v/v/v/v) HFIP/TFA/water/TES at RT for 45 min. The reactionmixture was diluted with 0.5 mL of water and all volatiles were removedunder a stream of nitrogen. N^(αB1)-conjugated insulin-linker conjugate9 was purified by RP-HPLC.

Yield: 4 mg (0.0007 mmol)

MS: m/z 1524.46=[M+4H]⁴⁺ (calculated=1524.75).

Example 10 Synthesis of N^(εB29)-Insulin Linker Conjugate 10

Insulin (644 mg, 0.111 mmol) was dissolved in 6.5 mL of DMSO. 3 mL ofcooled (4° C.) 0.5 M sodium borate buffer (pH 8.5) and 7f (70 mg, 0.073mmol) in 2.5 mL of DMSO were added and mixture was stirred for 5 min atRT. 400 μL AcOH were added and protected insulin conjugate was purifiedby RP HPLC.

Yield: 172 mg (0.025 mmol).

MS: m/z 1662.27=[M+4H]⁴⁺ (calculated=1662.48).

Removal of protecting groups was affected by treatment of lyophilizedproduct fractions with 6 mL of 90/10/2/2 (v/v/v/v) HFIP/TFA/TES/waterfor 1 h at RT. N^(αB29)-conjugated insulin-linker conjugate 10 waspurified by RP HPLC.

Yield: 143 mg (0.023 mmol).

MS: m/z 1531.46=[M+4H]⁴⁺ (calculated=1531.71).

Example 11 Preparation of Insulin-Linker-Hydrogel 11a, 11b, 11c, 11d,11da, 11 db, and 11dc

Dry maleimide functionalized hydrogel 4 (82 mg, 10.3 μmol maleimidogroups) was filled into a syringe equipped with a filter frit. Asolution of insulin-linker-thiol 8b (27.8 mg, 4.6 μmol) in 1.0 mLacetonitrile/water/TFA 1/1/0.001 (v/v/v) was added and the suspensionwas incubated for 5 min at RT. Acetate buffer (0.4 mL, pH 4.8, 1.0 M)was added and the sample was incubated at RT for 1 h. Consumption ofthiol was monitored by Ellman test. Hydrogel was washed 10 times with1/0.43/0.001 (v/v/v) acetonitrile/water/TFA and 2 times with1/1/0.2/0.25 (v/v/v/v) 1.0 M sarcosine pH 7.4/acetonitrile/0.5 Mphosphate buffer pH 7.4/water. Finally, the hydrogel was suspended inthe sarcosine solution and incubated for 2 h at RT.

Insulin-linker-hydrogel 11a was washed 10 times withacetonitrile/water/TFA 1/1/0.001 (v/v/v) and stored at 4° C.

Insulin content was determined by total hydrolysis of an aliquot ofinsulin-linker-hydrogel under reductive conditions at pH 12 andsubsequent insulin A-chain and insulin B-chain quantification byRP-HPLC.

Insulin loading of 11a: 175 mg insulin/g insulin-linker-hydrogel

Insulin amount in a 11a suspension in 10 mM sodium acetate buffer pH 5,135 mM sodium chloride: 12 mg insulin per 1 ml 11a suspension.

11b, 11c, and 11d were prepared as described above except for the use of8c, 9, and 10, respectively, instead of 8b.

11da was prepared as described above except for the use of 10 and 4ainstead of 8b and 4.

11 db was prepared as follows: A suspension of maleimide functionalizedhydrogel 4a in pH 2.5 HCl, 0.01% Tween-20 (5.0 mL, 119 μmol maleimidogroups) was filled into a syringe equipped with a filter. A solution ofinsulin-linker-thiol 10 (166 mg, 24.4 μmol) in 8.0 mL HCl pH 2.5, 0.01%Tween-20 was added and the suspension was incubated for 5 min at RT.Sodium succinate buffer (3.9 mL, pH 4.0, 150 mM; 1 mM EDTA, 0.01%Tween-20) was added to yield pH 3.6 and the sample was incubated at RTfor 90 min. Consumption of thiol was monitored by Ellman test. Hydrogelwas washed 10 times with sodium succinate buffer (pH 3.0, 50 mM; 1 mMEDTA, 0.01% Tween-20) and 3 times with sodium succinate buffer (pH 3.0,50 mM; 1 mM EDTA, 0.01% Tween-20) containing 200 mM acetyl cysteine.Finally, the hydrogel was suspended in the acetyl cysteine containingbuffer and incubated for 1 h at RT.

Insulin-linker-hydrogel 11 db was washed 10 times with succinate buffer(pH 3.0, 50 mM; 1 mM EDTA, 0.01% Tween-20) and 8 times with sodiumacetate buffer (pH 5.0, 10 mM; 130 mM NaCl, 0.01% Tween-20).

Insulin loading of 11 db: 6.12 mg insulin per mL insulin-linker-hydrogelsuspension

11dc was prepared as follows: A suspension of maleimide functionalizedhydrogel 4a in pH 2.5 HCl, 0.01% Tween-20 (58.3 mL, 958 μmol maleimidogroups) was added to a solid phase synthesis reactor. A solution ofinsulin-linker-thiol 10 (117 mL, 460 μmol) in 2.5 HCl, 0.01% Tween-20was added to 4a. The suspension was incubated at RT for 5 min. Succinatebuffer (4.8 mL, pH 4.0, 150 mM; 1 mM EDTA, 0.01% Tween-20) was added toyield a pH of 3.6 and the suspension was incubated at RT for 90 min.

Consumption of thiol was monitored by Ellman test. Hydrogel was washed10 times with succinate buffer (pH 3.0, 50 mM; 1 mM EDTA, 0.01%Tween-20) and 2 times with succinate buffer (pH 3.0, 50 mM; 1 mM EDTA,0.01% Tween-20) containing 10 mM mercaptoethanol. Finally, the hydrogelwas suspended in the mercaptoethanol containing buffer and incubated for3 h at RT.

Insulin-linker-hydrogel 11dc was washed 10 times with succinate buffer(pH 3.0, 50 mM; 1 mM EDTA, 0.01% Tween-20) and 6 times withsuccinate/Tris buffer (pH 5.0, 10 mM; 85 g/L trehalose, 0.01% Tween-20).

Insulin loading of 11dc: 18.7 mg insulin per mL insulin-linker-hydrogelsuspension

Alternatively, maleimide derivatized hydrogel microparticles 4aa can beused instead of 4a.

Example 12 Release Kinetics In Vitro

Insulin-linker-hydrogel 11a, 11b, 11c and 11d, respectively,(insulin-linker-hydrogel containing ca. 1 mg insulin) were suspended in2 ml 60 mM sodium phosphate, 3 mM EDTA, 0.01% Tween-20, pH 7.4, andincubated at 37° C. Suspensions were centrifuged at time intervals andsupernatant was analyzed by RP-HPLC at 215 nm and ESI-MS. UV-signalscorrelating to liberated insulin were integrated and plotted againstincubation time.

Curve-fitting software was applied to estimate the correspondinghalftime of release.

In vitro half-lives of 16 d, 10 d, 30 d, and 14 d were determined for11a, 11b, 11c and 11d, respectively.

Alternatively, insulin-linker-hydrogel 11 db was transferred to syringesequipped with filters, suspended in 6 ml 60 mM sodium phosphate, 3 mMEDTA, 0.01% Tween-20, pH 7.4, and incubated at 37° C. At defined timepoints the supernatant was exchanged and liberated insulin wasquantified by RP-HPLC at 215 nm. The amount of released insulin wasplotted against incubation time. Curve-fitting software was applied andan in vitro halftime of 15 d was determined for 11 db.

Alternatively, insulin-linker-hydrogel 11 db was filled in achromatography column and placed in a temperature controlled incubator(37° C.). Sodium phosphate (pH 7.4, 60 mM; 3 mM EDTA, 0.01% Tween-20)was pumped through the column with a constant flow of 0.25 mL/h(nominal) and collected outside the incubator. At defined time pointsthe solution was analyzed by RP-HPLC at 215 nm. The amount of releasedinsulin was plotted against incubation time. Curve-fitting software wasapplied and an in vitro halftime of 13 d was determined for 11 db.

Example 13 Synthesis of a LysB29-Linker Conjugate of Insulin (12a) and aLysB28-Linker Conjugate of Insulin Lispro (12b) Synthesis ofLysB29-Linker Conjugate of Insulin (12a)

1.2 g (0.206 mmol, 0.85 eq) of insulin were dissolved in DMSO at RT.After 30 min the solution was cooled to 0° C. while borate buffer (0.5M, pH 8.5, 21.6 ml) was added over a period of 4.40 min. The temperatureof the solution was kept between 25 and 28° C. A solution of 228 mg(0.239 mmol, 1 eq) 7f was dissolved in 40 ml DMSO was addedtime-invariant over a period of 3 min. The ice bath was removed and thereaction mixture was stirred for 5 min at RT. The reaction was quenchedby addition of 70 ml of MeCN/H₂O (1:1, 0.1% TFA) and 400 μl AcOH. 12awas purified by RP HPLC (solvent A: H₂O with 0.1% TFA, solvent B: MeCNwith 0.1% TFA, gradient: 30-80% B over 14 min, flow: 40 ml/min).

Regio-selectivity according to HPLC analysis (before RP HPLCpurification): 0.70% 7f attached to GlyA1 of insulin and 76.2% 7fattached to LysB29 of insulin (see FIG. 1 a).

Yield: 862 mg (TFA-salt, 60%).

MS [M+H]_(1/4) ⁺=1662.25 g/mol ((MW+H)_(1/4) calculated=1662.35 g/mol).

Synthesis of LysB28-Linker Conjugate of Insulin Lispro (12b)

0.347 g (0.059 mmol, 0.85 eq) of insulin lispro were dissolved in 6 mlDMSO at RT. After 30 min the solution was cooled to 0° C. while boratebuffer (0.5 M, pH 8.5, 5.64 ml) was added over a period of 1.40 min. Thetemperature of the solution was kept between 25 and 30° C. A solution of67 mg (0.070 mmol, 1 eq) 7f dissolved in 8 ml DMSO was addedtime-invariant over a period of 2 min. The ice bath was removed and thereaction mixture was stirred for 5 min at RT. The reaction was quenchedby addition of 20 ml of MeCN/H₂O (1:1, 0.1% TFA) and 1 ml AcOH. 12b waspurified by RP HPLC (solvent A: H₂O with 0.1% TFA, solvent B: MeCN with0.1% TFA, gradient: 30-80% B over 14 min, flow: 40 ml/min).

Regio-selectivity according to HPLC analysis (before RP HPLCpurification): 1.3% of the product was 7f attached to. GlyA1 of insulinlispro, 76.7% of the product was 7f attached to LysB28 of insulin lispro(see FIG. 1 b).

Yield: 305 mg (TFA-salt, 72%).

MS [M+H]_(1/4) ⁺=1662.25 g/mol ((MW+H)_(1/4) calculated=1662.35 g/mol).

Example 14 Pharmacokinetics Study in Rat

The pharmacokinetics of 11a were determined by measuring the plasmainsulin concentration after subcutaneous application of a single doseinto rats.

One group consisting of 10 male Wistar rats (200-250 g) was used tostudy the plasma insulin levels over a period of 14 days. Each of theanimals received a single subcutaneous injection of 500 μL 11asuspension in acetate buffer pH 5, containing 6 mg insulin (12 mginsulin/ml). Per animal and time point 200 μl of blood was withdrawnsublingually to obtain 100 μL Li-Heparin plasma. Samples were collectedbefore application and after 4 h, 1, 2, 3, 4, 7, 9, 11 and 14 days postinjection. Plasma samples were frozen within 15 min after bloodwithdrawal and stored at −80° C. until assayed.

Plasma insulin concentrations were measured using an ultrasensitiveInsulin ELISA kit (Mercodia) by following the manufacturer's protocol.Plasma samples were diluted in ELISA buffer (1:5 and 1:10 withcalibrator 0) prior to measurement. Insulin concentrations werecalculated from a calibration curve which was generated by measuringinsulin standards in duplicate and fitting the values using linearregression. The insulin concentration was defined as the mean from twoindependent dilution series corrected by the respective dilution factorand plotted against time. Averaged plasma insulin concentrations foreach time point were obtained by calculating the mean of all animalsused as shown in FIG. 2.

A bursless and sustained release of insulin over 14 days was observed.

Example 15 Pharmacokinetics Study in Rat

The pharmacokinetics of 11da were determined by measuring plasma insulinconcentrations over a period of 13 days in healthy rats.

8 Wistar rats (appr. 250 g body weight) received a single subcutaneousinjection of 500 μL of test item 11da in acetate buffer pH 5, containing3 mg insulin (approx. 12 mg/kg). Per animal and time point 200 μL ofblood was withdrawn from the tail vein to obtain about 100 μL Li-Heparinplasma. Samples were collected 1 day before and 4 h, 1 d, 2 d, 3 d, 6 d,7 d, 8 d, 10 d and 13 d after test item administration. Plasma sampleswere frozen and stored at −80° C. until assayed. The insulin content ofthe plasma samples was measured using a human insulin ultrasensitiveELISA Kit (DRG Instruments GmbH, Germany) following the manufacturer'sinstructions. Blanks (calibrator 0) were included in the calibrationcurve and were subtracted from the sample values and the calibrationcurve was fitted using a 3rd order polynomic equation. Before analysisplasma samples were vortexed and diluted in reaction tubes (1:5 and 1:10with calibrator 0). For analysis OD at 450 nm was measured with amicrotiter plate reader (Tecan Ultra) without reference wavelengthcorrection. Results of plasma insulin content up to day 13 for allanimals being investigated are shown in FIG. 3.

After a single subcutaneous injection of 500 μL 11d that contained 3 mginsulin the average plasma insulin level rose to a maximum of about 500pM on day 1. As expected the plasma insulin concentration subsequentlydecreased continuously within 2 weeks. The peak to trough ratio ofplasma insulin levels within the first week of the study wasapproximately 1.7

Example 16 Pharmacokinetics and Pharmacodynamics Study in Rats

The amount and the bioactivity of the released insulin was investigatedby analyzing the plasma insulin concentration and the blood glucoselowering effect in an exploratory pharmacokinetic/pharmacodynamic studyusing diabetic Sprague-Dawley (SD) rats.

For this purpose diabetes was induced in 8 rats with streptozotocin(STZ) and all animals with blood glucose levels above 350 mg/dL on day 0were included in this study. 7 out of 8 SD rats became diabetic andreceived a single subcutaneous injection of 500 μL test item 11da inacetate buffer pH 5, containing 6.4 mg insulin. Per animal and timepoint 200 μL of blood was withdrawn from the tail vein to obtain about100 μL Li-Heparin plasma. Samples were collected 4 days before and 2 h,1 d, 2 d, 3 d, 6 d, 7 d, 8 d, 10 d and 13 d after test itemadministration. Plasma samples were frozen and stored at −80° C. untilassayed. Blood glucose was measured with an AccuChek Comfort device fromthe tail vein 3 times before injection and 2 h, 1 d, 2 d, 3 d, 6 d, 7 d,8 d, 10 d, 13 d, 15 d, 17 d, 20 d, 22 d and 24 d after test itemadministration. The insulin content of the plasma samples was measuredusing a human insulin ELISA Kit (DRG Instruments GmbH, Germany)following the manufacturer's instructions. Blanks (calibrator 0) wereincluded in the calibration curve and were subtracted from the samplevalues and the calibration curve was fitted using a 3rd order polynomicequation. Before analysis plasma samples were vortexed and diluted inreaction tubes (1:5 and 1:10 with calibrator 0). For analysis OD at 450nm was measured with a microtiter plate reader (Tecan Ultra) withoutreference wavelength correction. The plasma insulin level was monitoredover 2 weeks and blood glucose level over a 3 week period as shown inFIG. 4.

After a single subcutaneous injection of insulin hydrogel 11da the bloodglucose level was effectively lowered over a period of 10 days withvalues below 100 mg/dL without any symptoms for hypoglycemia. Due to thehigher dosage of 6.4 mg insulin per animal, the maximal plasma insulinconcentration was approx. 800 pM on day 1 and decreased continuouslywithin 2 weeks to approx. 300 pM. Simultaneously the blood glucosevalues began to rise after 10 days and reached predose levels after 3weeks.

Example 17 Pharmacokinetics Study Over 24 Hours (Burst Study) in Rats

In order to prove that insulin is released from insulin-linker-hydrogelwithout a burst the plasma insulin concentration was monitored over aperiod of 24 hours in healthy rats.

8 Sprague-Dawley rats (200-250 g body weight) were divided into 2 groupsand received a single subcutaneous injection of 2 mL of test item 11 dbin acetate buffer pH 5 per kg body weight. The test item had aconcentration of 4 mg/mL insulin so that each animal received 8 mginsulin per kg body weight. Per animal and time point 200 μL of bloodwas withdrawn from the tail vein to obtain about 100 μL Li-Heparinplasma. Group A samples were collected predose and 5 min, 30 min, 2 h, 4h and 8 h after application of test item and for group B predose and 15min, 1 h, 3 h, 6 h and 24 h after test item administration. Plasmasamples were frozen and stored at −80° C. until assayed. The insulincontent of the plasma samples was measured using a human insulinultrasensitive ELISA Kit (DRG Instruments GmbH, Germany) following themanufacturer's instructions. Blanks (calibrator 0) were included in thecalibration curve and were subtracted from the sample values and thecalibration curve was fitted using a 3rd order polynomic equation.Before analysis plasma samples were vortexed and diluted in reactiontubes (1:5 and 1:10 with calibrator 0). For analysis OD at 450 nm wasmeasured with a microtiter plate reader (Tecan Ultra) without referencewavelength correction. The result is shown in FIG. 5 and clearlyindicates that insulin is released without any burst.

Example 18 Pharmacokinetics and Pharmacodynamics Multiple Dose Study inRats

The pharmacokinetics and pharmacodynamics after 3 weekly doses of 11dawere determined by measuring plasma insulin concentrations and bloodglucose levels over a period of 4 weeks in diabetic rats.

8 Sprague-Dawley rats were used with a mean body weight of 239 g.Diabetes was induced with streptozotocin (STZ) and all animals withblood glucose levels above 350 mg/dL on day 0 (test item injection day)were included in the study. 8 of 8 animals which received STZ treatmentbecame diabetic and received 3 weekly subcutaneous injections on day 0,7 and 14 of 2 mL test item 11da in acetate buffer pH 5 per kg bodyweight. With a test item insulin concentration of 4 mg/mL the applieddose was 8 mg insulin per kg body weight. Per animal and time point 200μL of blood was withdrawn from the tail vein to obtain about 100 μLLi-Heparin plasma. Samples were collected 3 days before and up to 28days after test item administration. Plasma samples were frozen andstored at −80° C. until assayed. Blood glucose was measured with anAccuChek Comfort device from the tail vein 3 times before injection andup to 30 days post injection. The insulin content of the plasma sampleswas measured using a human insulin ELISA Kit (DRG Instruments GmbH,Germany) following the manufacturer's instructions. Blanks (calibrator0) were included in the calibration curve and were subtracted from thesample values and the calibration curve was fitted using a 3rd orderpolynomic equation. Before analysis plasma samples were vortexed anddiluted in reaction tubes (1:5 and 1:10 with calibrator 0). For analysisOD at 450 nm was measured with a microtiter plate reader (Tecan Ultra)without reference wavelength correction. The plasma insulin level andthe blood glucose level were monitored over a 4 week period and are bothshown in FIG. 6.

The shape of the curves indicate that the released insulin was bioactiveby steadily lowering the blood glucose level to values about 100 mg/dLpost 3rd injection which remained low for about a week. At the same timethe maximal insulin concentration increased steadily starting from 200pM after the first and 300 pM after the second dose to 400 pM followingthe third dose and subsequently decreased again within 2 weeks to valuesbelow 100 pM.

Example 19 Pharmacokinetics Study in Rat

The pharmacokinetics of 11dc were determined by measuring plasma insulinconcentrations over a period of 13 days in healthy rats.

8 Wistar rats (appr. 230 g body weight) received a single subcutaneousinjection of 2 ml/kg of test item 11dc in succinate buffer pH 5 (10 mMsuccinate/tris, 85 g/l trehalose, 0.01% Tween-20, pH 5.0), containing 3mg insulin (12 mg/kg dose). Per animal and time point 200 μL of bloodwas withdrawn from the tail vein to obtain about 100 μL Li-Heparinplasma. Samples were collected 4 days before and 0.3 h (4 animals), 1 h(4 animals), 2 h (4 animals), 4 h (4 animals), 1 d, 2 d, 3 d, 6 d, 8 d,10 d and 13 d after test item administration. Plasma samples were frozenand stored at −80° C. until assayed. The insulin content of the plasmasamples was measured using a human insulin ultrasensitive ELISA Kit (DRGInstruments GmbH, Germany) following the manufacturer's instructions.Blanks (calibrator 0) were included in the calibration curve and weresubtracted from the sample values and the calibration curve was fittedusing a 3rd order polynomic equation. Before analysis plasma sampleswere vortexed and diluted in reaction tubes (1:5 and 1:10 withcalibrator 0). For analysis OD at 450 nm was measured with a microtiterplate reader (Tecan Ultra) without reference wavelength correction.Results of plasma insulin content up to day 13 for all animals beinginvestigated are shown in FIG. 7.

After a single subcutaneous injection of 12 mg/kg 11dc the averageplasma insulin level rose to a maximum of about 500 pM on day 1. Asexpected the plasma insulin concentration subsequently decreasedcontinuously within 2 weeks. The peak to trough ratio of plasma insulinwithin the first week was approximately 1.4.

Example 20 Real-Time Insulin Release and Hydrogel Degradation at pH 7.4

Insulin-linker hydrogel 11a (730 μL, containing 3.19 mg insulin) in pH5.0 acetate buffer (10 mM, 130 mM NaCl, 0.01% (w/v) tween-20) was filledin a sample preparation tube, washed 3× with pH 7.4 release buffer (60mM sodium phosphate, 3 mM EDTA, 0.01% (w/v) Tween-20) and filled-up to1.00 mL. An Aliquot of the suspension (0.5 mL, 1.59 mg insulin) wasfilled in a chromatography column and placed in a temperature controlledincubator (37° C.). Release buffer (pH 7.4) was pumped through thecolumn with a constant flow of 0.25 mL/h (nominal) and collected outsidethe incubator. At defined time points the solution was analyzed byRP-HPLC (215 nm). The amount of released insulin was plotted againstincubation time and curve-fitting software was applied to estimate thecorresponding halftime of release. A halftime of 9.4 d for the insulinrelease was determined.

After 39 d incubation at 37° C. the hydrogel suspension was transferredto a sample preparation tube, residual hydrogel was washed out of thecolumn with pH 7.4 release buffer and the sample was filled-up to 1.00mL. Two aliquots (300 μL each) were transferred to sterile samplepreparation tubes, filled-up to 1.5 mL, and incubated at 37° C. Sampleswere taken at time intervals and analyzed by size exclusionchromatography. UV-signals corresponding to hydrogel releasedwater-soluble degradation products comprising one or more backbonemoieties (corresponding to reactive functional groups) were integratedand plotted against incubation time, see FIG. 8.

Example 21 Injectability of Insulin-Linker-Hydrogel Prodrug

5 mL insulin-linker-hydrogel prodrug 11dc (bead size distribution from32-75 μm, 18 mg insulin/ml) in pH 5.0 succinic acid/tris (10 mM, 40 g/Lmannitol; 10 g/L trehalose dihydrate; 0.05% TWEEN-20) was used. Theinsulin-linker-hydrogel prodrug suspension was filled into a 1 mLsyringe (length 57 mm) via a 20 G needle. The 20 G needle was replacedby a 30 G needle and placed into the syringe mounting (Aqua ComputerGmbH&Co. KG) and the measurement was started with a piston velocity of172 mm/min (equals 50 μL/s) (Force test stand: Multitest 1-d, Datarecording software: EvaluatEmperor Lite, Version 1.16-015, Forge Gauge:BFG 200 N (all Mecmesin Ltd., UK). Experiments with increasing pistonvelocities shown in the table below were carried out with a newinsulin-linker-hydrogel prodrug sample. The experiments with water andethylene glycol were carried out accordingly. For all of the experimentsthe same 30 G needle was used. Force versus flow using a 30 G needle isshown in FIG. 9.

Velocity of Flow/ Flow/ piston/ Force/N Force/N Force/N (sec/mL)(μL/sec) (mm/min) (water) 11dc (ethylene glycol) 6 167 573 13 36 83 8125 430 10 29 62 10 100 344 7 24 51 15 67 229 4 22 35 20 50 172 3 17 27

ABBREVIATIONS

-   AcOH acetic acid-   AcOEt ethyl acetate-   Aib 2-Aminoisobutyric acid-   Bn benzyl-   Boc t-butyloxycarbonyl-   COMU    (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium    hexafluorophosphate-   DBU 1,3-diazabicyclo[5.4.0]undecene-   DCC N,N,-dicyclohexylcarbodiimid-   DCM dichloromethane-   DIEA diisopropylethylamine-   DMAP dimethylamino-pyridine-   DMF N,N-dimethylformamide-   Dmob 2,4-dimethoxybenzyl-   DMSO dimethylsulfoxide-   EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimid-   EDTA ethylenediaminetetraacetic acid-   eq stoichiometric equivalent-   ESI-MS electrospray ionization mass spectrometry-   EtOH ethanol-   Fmoc 9-fluorenylmethoxycarbonyl-   HATU O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HFIP hexafluoroisopropanol-   HPLC high performance liquid chromatography-   HOBt N-hydroxybenzotriazole-   iPrOH 2-propanol-   LCMS mass spectrometry-coupled liquid chromatography-   Mal 3-maleimido propyl-   Mal-PEG6-NHS    N-(3-maleimidopropyl)-21-amino-4,7,10,13,16,19-hexaoxa-heneicosanoic    acid NHS ester-   Me methyl-   MeCN acetonitrile-   MeOH methanol-   Mmt 4-methoxytrityl-   MS mass spectrum/mass spectrometry-   MTBE methyl tert.-butyl ether-   MW molecular mass-   n.d. not determined-   NHS N-hydroxy succinimide-   OD optical density-   OBu butyloxy-   OtBu tert.-butyloxy-   PEG poly(ethylene glycol)-   Phth phthal--   PyBOP benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium    hexafluorophosphate-   RP-HPLC reversed-phase high performance liquid chromatography-   rpm rounds per minute-   RT room temperature-   SEC size exclusion chromatography-   Su succinimidyl-   TCP 2-chlorotrityl chloride resin-   TES triethylsilane-   TFA trifluoroacetic acid-   THF tetrahydrofurane-   TMEDA N,N,N′N′-tetramethylethylene diamine-   Tmob 2,4,6-trimethoxybenzyl-   Trt triphenylmethyl, trityl-   HPLC ultra performance liquid chromatography-   UV ultraviolet-   VIS visual

1. A prodrug or a pharmaceutically acceptable salt thereof comprising aninsulin linker conjugate D-L, wherein D represents the insulin moiety;and -L is a non-biologically active linker moiety -L¹ represented byformula (I),

wherein the dashed line indicates the attachment to one of the aminogroups of the insulin by forming an amide bond; X is C(R³R^(3a)); orN(R³); R^(1a), R^(3a) are independently selected from the groupconsisting H, NH(R^(2b)), N(R^(2b))C(O)R⁴ and C₁₋₄ alkyl; R¹, R²,R^(2a), R^(2b), R³, R⁴ are independently selected from the groupconsisting of H and C₁₋₄ alkyl, wherein L¹ is substituted with one L²-Zand optionally further substituted, provided that the hydrogen markedwith the asterisk in formula (I) is not replaced by a substituent andwherein L² is a single chemical bond or a spacer; and Z is a hydrogel.2. Prodrug of claim 1, wherein in formula (I) R² is replaced by L²-Z. 3.Prodrug of claim 1, wherein in formula (I) R¹ is replaced by L²-Z. 4.Prodrug of any of claims 1 to 3, wherein in formula (I) X is N(R³). 5.Prodrug of any of claims 1 to 3, wherein in formula (I) X is C(R³R^(3a))and R^(3a) is N(R^(2b))C(O)R⁴.
 6. Prodrug of claim 1, wherein X isC(R³R^(3a)) and R^(3a) is replaced by L²-Z.
 7. Prodrug of claim 1,wherein X is C(R³R^(3a)), R^(3a) is N(R^(2b))-L²-Z.
 8. Prodrug of any ofclaims 1 to 7, wherein L¹ is not further substituted.
 9. Prodrug of anyof claims 1 to 8, wherein the insulin moiety is attached to L¹ throughthe nitrogen N^(αA1).
 10. Prodrug of any of claims 1 to 9, whereininsulin is recombinant human insulin.
 11. Prodrug of claim 10, whereinthe recombinant human insulin is attached to L¹ through the nitrogen ofa lysine side chain of the insulin moiety.
 12. Prodrug of any of claims1 to 11, wherein the hydrogel Z is a biodegradable poly(ethylene glycol)(PEG) based water-insoluble hydrogel.
 13. Prodrug of any of claims 1 to12, wherein the hydrogel is composed of backbone moieties interconnectedby hydrolytically degradable bonds.
 14. Prodrug of claim 13, wherein thebackbone moieties have a molecular weight in the range of from 1 kDa to20 kDa.
 15. Prodrug of claim 13 or 14, wherein backbone moieties arelinked together through crosslinker moieties, each crosslinker moietybeing terminated by at least two of the hydrolytically degradable bonds.16. Prodrug of claim 15, wherein the crosslinker moieties have amolecular weight in the range of from 0.5 kDa to 5 kDa.
 17. Prodrug ofclaims 13 to 16, wherein L² is connected to a backbone moiety.
 18. Theprodrug of any of claims 1 to 17, wherein L² is attached to Z via aterminal group having the following structure Error! Objects cannot becreated from editing field codes, wherein the dashed lines indicate theattachment to L² and Z, respectively.
 19. Prodrug of claim 1, of formula(IIa)

wherein N^(ε)-Insulin refers to insulin connected via one lysine sidechain.
 20. Prodrug of claim 1 or 2, of formula (IIb)


21. The prodrug of claim 19 or 20, wherein the hydrogel is abiodegradable polyethylene glycol (PEG) based water-insoluble hydrogel.22. The prodrug of any of claims 19 to 21, wherein the hydrogel iscomposed of backbone moieties interconnected by hydrolyticallydegradable bonds.
 23. The prodrug of any of claims 13 to 18, 22, whereinthe backbone moieties comprise a branching core of the followingformula:

wherein the dashed line indicates attachment to the remainder of thebackbone moiety.
 24. The prodrug of any of claims 13 to 18, 22, 23wherein the backbone moieties comprise a structure of the followingformula:

wherein n is an integer of from 5 to 50 and the dashed line indicatesattachment to the rest of the molecule.
 25. The prodrug of any of claims13 to 24, wherein the backbone moiety comprises a hyperbranched moietyHyp.
 26. The prodrug of claim 25, wherein the backbone moiety comprisesa hyperbranched moiety Hyp of the following formula:

wherein the dashed lines indicate attachment to the rest of the moleculeand carbon atoms marked with asterisks indicate S-configuration.
 27. Theprodrug of any of claims 22 to 26, wherein the backbone moieties areattached to at least one spacer of the following formula:

wherein one of the dashed lines indicates attachment to thehyperbranched moiety Hyp and the second dashed line indicates attachmentto the rest of the molecule; and wherein m is an integer of from 2 to 4.28. The prodrug of any of claim 22 or 27, wherein the backbone moietiesare attached to at least one spacer of the following formula:

wherein the dashed line marked with the asterisk indicates the bondbetween the hydrogel and the N of the thiosuccinimide group of claim 18,wherein the other dashed line indicates attachment to Hyp, and wherein pis an integer of from 0 to
 10. 29. The prodrug of any of claims 15 to28, wherein the backbone moieties are linked together throughcrosslinker moieties comprising the following structure

wherein q is an integer from 3 to
 100. 30. The prodrug of any of claims1 to 29 in the form of microparticles.
 31. The prodrug of claim 30,wherein the microparticles have a diameter of between 20 and 100micrometer
 32. The prodrug of claim 30, wherein the microparticles canbe administered by injection through a needle smaller than 0.6 mm innerdiameter.
 33. The prodrug of claim 30, wherein the microparticles can beadministered by injection through a needle smaller than 0.3 mm innerdiameter.
 34. The prodrug of claim 30, wherein the microparticles can beadministered by injection through a needle smaller than 0.2 mm innerdiameter.
 35. An insulin-linker conjugate of formula (IIIa)

wherein N^(ε)-Insulin refers to insulin connected via one lysine sidechain.
 36. An insulin-linker conjugate of formula (IIIb)


37. A pharmaceutical composition comprising a prodrug of any of claims 1to 34 or a pharmaceutically acceptable salt thereof together with apharmaceutically acceptable excipient.
 38. A pharmaceutical compositionaccording to claim 37, wherein the pharmaceutical composition is dry.39. A pharmaceutical composition according to claim 38, wherein thepharmaceutical composition is dried by lyophilization.
 40. A compositionaccording to any of claims 37 to 39, wherein the insulin hydrogelprodrug is sufficiently dosed in the composition to provide atherapeutically effective amount of insulin for at least three days inone application.
 41. A composition according to any of claims 37 to 40,wherein it is a single dose composition.
 42. A composition according toany of claims 37 to 40, wherein it is a multiple dose composition.
 43. Acomposition according to any of claims 37 to 42, wherein it contains oneor more additional biologically active agents, either in their freeform; as prodrugs, especially as hydrogel prodrugs; and wherein the oneor more additional biologically active agents are selected from thegroup consisting of sulfonylureas, such as, for example, chlorpropamide,tolazamide, tolbutamide, glyburide, glipizide, glimepiride, and thelike; meglitinides, such as, for example, repaglinide; glucagon-likepeptide-1 (GLP-1) and its mimetics, glucose-insulinotropic peptide (GIP)and it's mimetics, exendin and its mimetics, and dipeptyl proteaseinhibitors (DPPIV); biguanides, such as, for example, metformin;thiazolidinediones, such as, for example, rosiglitazone, pioglitazone,troglitazone, isaglitazone (known as MCC-555),2-[2-[(2R)-4-hexyl-3,4-dihydro-3-oxo-2H-1,4-benzoxazin-2-yl]ethoxy]-benzeneacetic acid, and the like; GW2570, and the like; retinoid-X receptor(RXR) modulators, such as, for example, targretin, 9-cis-retinoic acid,and the like; other insulin sensitizing agents, such as, for example,INS-1, PTP-1B inhibitors, GSK3 inhibitors, glycogen phosphorylase ainhibitors, fructose-1,6-bisphosphatase inhibitors, and the like;insulins, including regular or short-acting, intermediate-acting, andlong-acting insulins, inhaled insulin and insulin analogues, such asinsulin molecules with minor differences in the natural amino acidsequence; small molecule mimics of insulin, including, but not limitedto L-783281, TE-17411, and the like; Na-glucose co-transporterinhibitors, such as T-1095, T-1095A, phlorizen, and the like; amylinagonists which include, but are not limited to pramlintide, and thelike; glucagon antagonists such as AY-279955, and the like; anti-obesityagents such as orlistat, a pancreatic lipase inhibitor; sibutramine;norepinephrine and dopamine; growth hormone, IGF-1, growth hormonereleasing factor; oxyntomodulin and ghrelin modulators;appetite-suppressants such as benzphetamine, phenmetrazine, phentermine,diethylpropion, mazindol, sibutramine, phenylpropanolamine or,ephedrine; appetite-suppressants such as quipazine, fluoxetine,sertraline, fenfluramine, or dexfenfluramine; appetite-suppressantagents such as apomorphine; appetite-suppressant such as histaminemimetics, H3 receptor modulators; enhancers of energy expenditure suchas beta-3 adrenergic agonists and stimulators of uncoupling proteinfunction; leptin and leptin mimetics (eg, metreleptin); neuropeptide Yantagonists; melanocortin-1, 3 and 4 receptor modulators;cholecystokinin agonists; glucagon-like peptide-1 mimetics and analogueslike for example exendin; androgens such as dehydroepiandrosterone andderivatives such as etiocholandione; testosterone; anabolic steroidssuch as oxandrolone, and steroidal hormones; galanin receptorantagonists; cytokine agents such as ciliary neurotrophic factor;amylase inhibitors.
 44. A container comprising the pharmaceuticalcomposition according to claims 37 to
 43. 45. A container according toclaim 44, wherein the container is a dual-chamber syringe.
 46. Asuspension comprising the pharmaceutical composition according to claims37 and 40 to
 43. 47. A method of preparing a suspension according toclaim 46, comprising the steps of reconstituting the dry pharmaceuticalcomposition according to claim 38 or 39 by adding reconstitutionsolution.
 48. A kit of parts comprising a needle and a containercontaining reconstitution solution and the dry composition according toclaim 38 or 39 for use with the needle.
 49. A kit of parts according toclaim 48, wherein the container is a dual-chamber syringe and whereinone of the two-chambers of the dual-chamber syringe contains the drypharmaceutical composition and the second chamber of said dual-chambersyringe contains the reconstitution solution.
 50. A prodrug of any ofclaims 1 to 34 or a pharmaceutical composition of any of claims 37 to 43for use as a medicament.
 51. A prodrug of any of claims 1 to 34 or apharmaceutical composition of any of claims 37 to 43 for use in a methodof treating or preventing diseases or disorders which can be treated byinsulin.
 52. A process for acylating the ε-amino group of insulin or aninsulin analog, having one or more free α-amino groups and the freeε-amino group with an acylating agent containing one or more protectedfunctional groups, which comprises reacting the insulin or insulinanalog with a soluble acylating agent containing one or more protectedfunctional groups at a pH of 8.0 to below 9.0 in a polar solvent. 53.The process of claim 52, wherein the pH is from 8.0 to 8.9.
 54. Aninsulin compound characterized by having an acyl group linked to theε-nitrogen of insulin or an insulin analog and wherein such acyl grouphas one or more protected functional groups.
 55. An insulin-linkerreagent D-L*, wherein D represents an insulin moiety; and L* is anon-biologically active linker reagent represented by formula (IV),

wherein the dashed line indicates the attachment to one of the aminogroups of the insulin by forming an amide bond; X is C(R³R^(3a)); orN(R³); R^(1a), R^(3a) are independently selected from the groupconsisting H, NH(R^(2b)), N(R^(2b))C(O)R⁴ and C₁₋₄ alkyl; R¹, R²,R^(2a), R^(2b), R³, R⁴ are independently selected from the groupconsisting of H and C₁₋₄ alkyl, wherein L* is substituted with one L²*and optionally further substituted, provided that the hydrogen markedwith the asterisk in formula (IV) is not replaced by a substituent andwherein L²* is a spacer connected to L* and comprising a chemicalfunctional group intended for conjugation to a reactive biodegradablehydrogel;
 56. The insulin-linker reagent of claim 55, wherein in formula(IV) R² is replaced by L²*.
 57. The insulin-linker reagent of claim 56,wherein in formula (IV) R¹ is replaced by L²*.
 58. The insulin-linkerreagent of any of claims 55 to 57, wherein in formula (IV) X is N(R³).59. The insulin-linker reagent of any of claims 55 to 57, wherein informula (IV) X is C(R³R^(3a)) and R^(3a) is N(R^(2b))C(O)R⁴.
 60. Theinsulin-linker reagent of claim 55, wherein X is C(R³R^(3a)) and R^(3a)is replaced by L²*.
 61. The insulin-linker reagent of claim 55, whereinX is C(R³R^(3a)), R^(3a) is N(R^(2b))-L²*.
 62. The insulin-linkerreagent of any of claims 55 to 61, wherein L* is not furthersubstituted.
 63. The insulin-linker reagent of any of claims 55 to 62,wherein L²* comprises a thiol group.
 64. The insulin-linker reagent ofany of claims 55 to 62, wherein L²* comprises a maleimide group.
 65. Aprocess for the preparation of a prodrug of any of claims 1 to 34 and55, 51 comprising the steps of (a) contacting an aqueous suspensioncomprising maleimide-functionalized hydrogel microparticles with asolution comprising the insulin-linker reagent of claim 63 attemperatures between room temperature and 4° C. in a buffered aqueoussolution of pH 2-5, resulting in an insulin-linker-hydrogel conjugate;(b) optionally, treating the insulin-linker-hydrogel conjugate from step(a) with a thiol-containing compound of 34 Da to 500 Da at temperaturesbetween room temperature and 4° C. in a buffered aqueous solution of pH2-5.
 66. A process for the preparation of a prodrug of any of claims 1to 34 and 55, 51 comprising the steps of (a) contacting an aqueoussuspension comprising thiol-functionalized hydrogel microparticles witha solution comprising the insulin-linker reagent of 64 at temperaturesbetween room temperature and 4° C. in a buffered aqueous solution of pH2-5, resulting in an insulin-linker-hydrogel conjugate; (b) optionally,treating the insulin-linker-hydrogel conjugate from step (a) with amaleimide-containing compound of 100 to 300 Da at temperatures betweenroom temperature and 4° C. in a buffered aqueous solution of pH 2-5. 67.A process for preparing a needle injectable prodrug comprising the stepof (a) preparing a prodrug of claim 30 in the form of microparticles;(b) sieving the microparticles (c) selecting a fraction with a prodrugbead diameter of between 25 and 80 μm. (d) suspending the bead fractionof step (c) in an aqueous buffer solution suitable for injection. 68.Needle injectable prodrug obtainable from the process of claim
 67. 69.Prodrug of claim 68, injectable through a needle with an inner diameterof less than 300 μm.
 70. Prodrug of claim 68, injectable through aneedle with an inner diameter of less than 225 μm.
 71. Prodrug of claim68, injectable through a needle with an inner diameter of less than 175μm.